Humanized antibodies

ABSTRACT

The invention relates to humanized antibodies that bind to an epitope at the N-terminus of pyroglutamated amyloid beta (Aβ N3pE) peptide and to preventive and therapeutic treatment of diseases and conditions that are related to accumulation and deposition of amyloid peptides, such as amyloidosis, a group of disorders and abnormalities associated with pyroglutamated amyloid peptide, like Alzheimer&#39;s disease, Down&#39;s syndrome, cerebral amyloid angiopathy and other related aspects. More specifically, it pertains to the use of humanized monoclonal antibodies to bind pyroglutamated amyloid beta peptide in plasma, brain, and cerebrospinal fluid to prevent accumulation or to reverse deposition of Aβ N3pE within the brain and in various tissues in the periphery, and to alleviate amyloidosis. The present invention further pertains to diagnostic assays for the diagnosis of amyloidosis using the humanized antibodies of the invention.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of PCT/EP2016/066924, filedon Jul. 15, 2016, and claims the benefit of U.S. Provisional ApplicationNo. 62/209,650, filed on Aug. 25, 2015, and U.S. Provisional ApplicationNo. 62/193,356, filed on Jul. 16, 2015, each of which is incorporatedherein by reference.

FIELD OF THE INVENTION

The invention relates to humanized antibodies that bind to an epitope atthe N-terminus of pyroglutamated amyloid beta (Aβ N3pE) peptide and topreventive and therapeutic treatment of diseases and conditions that arerelated to accumulation and deposition of amyloid peptides, such asamyloidosis, a group of disorders and abnormalities associated withpyroglutamated amyloid peptide, like Alzheimer's disease, Down'ssyndrome, cerebral amyloid angiopathy and other related aspects. Morespecifically, it pertains to the use of humanized monoclonal antibodiesto bind pyroglutamated amyloid beta peptide in plasma, brain, andcerebrospinal fluid to prevent accumulation or to reverse deposition ofAβ N3pE within the brain and in various tissues in the periphery, and toalleviate amyloidosis. The present invention further pertains todiagnostic assays for the diagnosis of amyloidosis using the humanizedantibodies of the invention.

Sequence Listing Incorporation Biological sequence information for thisapplication is included in an ASCII text file, filed with theapplication, having the file name “MAI211SEQ.txt”, created on Jan. 10,2018, and having a file size of 40,710, which is incorporated herein byreference.

BACKGROUND ART

Amyloidosis is not a single disease entity but rather a diverse group ofprogressive disease processes characterized by extracellular tissuedeposits of a waxy, starch-like protein called amyloid, whichaccumulates in one or more organs or body systems. As the amyloiddeposits accumulate, they begin to interfere with the normal function ofthe organ or body system. There are at least 15 different types ofamyloidosis. The major forms are primary amyloidosis without knownantecedent, secondary amyloidosis following some other condition, andhereditary amyloidosis.

Secondary amyloidosis occurs during chronic infection or inflammatorydisease, such as tuberculosis, a bacterial infection called familialMediterranean fever, bone infections (osteomyelitis), rheumatoidarthritis, inflammation of the small intestine (granulomatous ileitis),Hodgkin's disease and leprosy.

Amyloid deposits include amyloid P (pentagonal) component (AP), aglycoprotein related to normal serum amyloid P (SAP), and sulphatedglycosaminoglycans (GAG), complex carbohydrates of connective tissue.Amyloid protein fibrils, which account for about 90% of the amyloidmaterial, comprise one of several different types of proteins. Theseproteins are capable of folding into so-called “beta-pleated” sheetfibrils, a unique protein configuration which exhibits binding sites forCongo red resulting in the unique staining properties of the amyloidprotein.

Many diseases of aging are based on or associated with amyloid-likeproteins and are characterized, in part, by the buildup of extracellulardeposits of amyloid or amyloid-like material that contribute to thepathogenesis, as well as the progression of the disease. These diseasesinclude, but are not limited to, neurological disorders such as mildcognitive impairment (MCI), Alzheimer's disease (AD), like for instancesporadic Alzheimer's disease (SAD) or Familial Alzheimer's dementias(FAD) like Familial British Dementia (FBD) and Familial Danish Dementia(FDD), neurodegeneration in Down Syndrome, Lewy body dementia,hereditary cerebral hemorrhage with amyloidosis (Dutch type); the GuamParkinson-Dementia complex. Other diseases which are based on orassociated with amyloid-like proteins are progressive supranuclearpalsy, multiple sclerosis; Creutzfeld Jacob disease, Parkinson'sdisease, HIV-related dementia, ALS (amyotropic lateral sclerosis), AdultOnset Diabetes; senile cardiac amyloidosis; endocrine tumors, andothers, including macular degeneration.

Although pathogenesis of these diseases may be diverse, theircharacteristic deposits often contain many shared molecularconstituents. To a significant degree, this may be attributable to thelocal activation of pro-inflammatory pathways thereby leading to theconcurrent deposition of activated complement components, acute phasereactants, immune modulators, and other inflammatory mediators (McGeeret al., Tohoku J Exp Med. 174(3): 269-277 (1994)).

Recently, accumulating evidence demonstrates involvement of N-terminalmodified Aβ peptide variants in Alzheimer's disease. Aiming biopsiesdisplay a presence of Aβ 1-40 and Aβ 1-42 not only in the brain ofAlzheimer's patients but also in senile plaques of unaffectedindividuals. However, N-terminal truncated and pyroGlu modified AβN3pE-40/Aβ N3pE-42 is almost exclusively engrained within plaques ofAlzheimer's disease patients, making this Aβ variant an eligiblediagnostic marker and a potential target for drug development.

At present, several commercial manufacturers offer ELISA kits whichallow a detection of Aβ 1-40/1-42 and Aβ N3pE-40/Aβ N3pE-42 in the lowpicogram (pg) range.

The brains of Alzheimer's disease (AD) patients are morphologicallycharacterized by the presence of neurofibrillary tangles and by depositsof Aβ peptides in neocortical brain structures (Selkoe, D. J. & Schenk,D. Alzheimer's disease: molecular understanding predicts amyloid-basedtherapeutics. Annu. Rev. Pharmacol. Toxicol. 43, 545-584 (2003)). Aβpeptides are liberated from the amyloid precursor protein (APP) aftersequential cleavage by β- and γ-secretase. The γ-secretase cleavageresults in the generation of Aβ 1-40 and Aβ 1-42 peptides, which differin their C-termini and exhibit different potencies of aggregation,fibril formation and neurotoxicity (Shin, R. W. et al. Amyloidbeta-protein (Abeta) 1-40 but not Abeta 1-42 contributes to theexperimental formation of Alzheimer disease amyloid fibrils in ratbrain. J. Neurosci. 17, 8187-8193 (1997); Iwatsubo, T. et al.Visualization of Abeta 42(43) and Abeta 40 in senile plaques withend-specific Abeta monoclonals: evidence that an initially depositedspecies is Abeta 42(43). Neuron 13, 45-53 (1994); Iwatsubo, T., Mann, D.M., Odaka, A., Suzuki, N. & Ihara, Y. Amyloid beta protein (Abeta)deposition: Abeta 42(43) precedes Abeta 40 in Down syndrome. Ann.Neurol. 37, 294-299 (1995); Hardy, J. A. & Higgins, G. A. Alzheimer'sdisease: the amyloid cascade hypothesis. Science 256, 184-185 (1992);Roßner, S., Ueberham, U., Schliebs, R., Perez-Polo, J. R. & Bigl, V. Theregulation of amyloid precursor protein metabolism by cholinergicmechanisms and neurotrophin receptor signaling. Prog. Neurobiol. 56,541-569 (1998)).

The majority of Aβ peptides deposited in diffuse plaques are N-terminaltruncated or modified. Studies of Piccini and Saido have shown that thecore structure of senile plaques and vascular deposits consist of 50%pyroglutamate (pyroGlu) modified peptides (Piccini et al., J Biol Chem.2005 Oct. 7; 280(40):34186-92; Saido et al., Neuron. 1995 February;14(2): 457-66). PyroGlu modified peptides are more strongly cytotoxicthan other Aβ species and stable against aminopeptidases (Russo et al.,J Neurochem. 2002 September; 82(6):1480-9). Thus, pyroGlu Aβ specieshave a longer half-life whereby the accumulation of these species andthe formation of neurotoxic oligomers as well as aggregates arebeneficial (Saido, Neurobiol Aging. 1998 January-February; 19(1Suppl):S69-75). Due to the cyclization of glutamate to pyroGlu, chargedamino acids will be lost which strongly reduces the solubility of thepeptide and causes an increased aggregation tendency. In vitro studieshave shown that the initial oligomerisation of e.g. Aβ3(pE) is muchfaster compared to non-modified peptides (Schilling et al.,Biochemistry. 2006 Oct. 17; 45(41):12393-9). The Aβ N3pE-42 peptidescoexist with Aβ 1-40/1-42 peptides (Saido, T. C. et al. Dominant anddifferential deposition of distinct beta-amyloid peptide species, AbetaN3pE, in senile plaques. Neuron 14, 457-466 (1995); Saido, T. C., Yamao,H., Iwatsubo, T. & Kawashima, S. Amino- and carboxyl-terminalheterogeneity of beta-amyloid peptides deposited in human brain.Neurosci. Lett. 215, 173-176 (1996)), and, based on a number ofobservations, could play a prominent role in the pathogenesis of AD. Forexample, a particular neurotoxicity of Aβ N3pE-42 peptides has beenoutlined (Russo, C. et al. Pyroglutamate-modified amyloidbeta-peptides—AbetaN3(pE)—strongly affect cultured neuron and astrocytesurvival. J. Neurochem. 82, 1480-1489 (2002) and the pE-modification ofN-truncated Aβ peptides confers resistance to degradation by mostaminopeptidases as well as Aβ-degrading endopeptidases (Russo, C. et al.Pyroglutamate-modified amyloid beta-peptides—AbetaN3(pE)—strongly affectcultured neuron and astrocyte survival. J. Neurochem. 82, 1480-1489(2002); Saido, T. C. Alzheimer's disease as proteolytic disorders:anabolism and catabolism of beta-amyloid. Neurobiol. Aging 19, S69-S75(1998)). The cyclization of glutamic acid into pE leads to a loss ofN-terminal charge resulting in accelerated aggregation of Aβ N3pEcompared to the unmodified Aβ peptides (He, W. & Barrow, C. J. The Abeta3-pyroglutamyl and 11-pyroglutamyl peptides found in senile plaque havegreater beta-sheet forming and aggregation propensities in vitro thanfull-length A beta. Biochemistry 38, 10871-10877 (1999); Schilling, S.et al. On the seeding and oligomerization of pGlu-amyloid peptides (invitro). Biochemistry 45, 12393-12399 (2006)). Thus, reduction of AβN3pE-42 formation should destabilize the peptides by making them moreaccessible to degradation and would, in turn, prevent the formation ofhigher molecular weight Aβ aggregates and enhance neuronal survival.

However, for a long time it was not known how the pE-modification of Aβpeptides occurs. Recently, it was discovered that glutaminyl cyclase(QC) is capable to catalyze Aβ N3pE-42 formation under mildly acidicconditions and that specific QC inhibitors prevent Aβ N3pE-42 generationin vitro (Schilling, S., Hoffmann, T., Manhart, S., Hoffmann, M. &Demuth, H.-U. Glutaminyl cyclases unfold glutamyl cyclase activity undermild acid conditions. FEBS Lett. 563, 191-196 (2004); Cynis, H. et al.Inhibition of glutaminyl cyclase alters pyroglutamate formation inmammalian cells. Biochim. Biophys. Acta 1764, 1618-1625 (2006)).

All facts suggest that pyroGlu Aβ is a kind of germ for theinitialization of fibril formation. In a further study (Piccini et al.,2005, supra) volunteers with plaque depositions but without AD specificpathology could be distinguished from AD patients due to thecharacteristic amount of Aβ-species. Thereby the amount of N-terminaltruncated, pyroGlu modified peptides was significant higher in the brainof AD patients.

The posttranslational formation of pyroGlu at position 3 or 11 ofAβ-peptide implies cyclization of an N-terminal glutamate residue.Glutaminyl cyclase (QC) plays an important role in the generation ofpyroGlu peptides. QC is wide-spread in the plant- and animal kingdom andinter alia, is involved in the maturation of peptide hormones. Both thecyclisation of glutamine by release of ammonia and of glutamate byrelease of water to pyroGlu is performed by QC. In contrast to theglutamine cyclization the glutamate cyclisation occurs notspontaneously. QC catalyses the efficient (unwanted) side reaction fromglutamate to pyroGlu. The generated pyroGlu residue protects the proteinagainst proteolytic degradation. There are several references whichshows that QC plays an important role in the generation of pyroGlu Aβ:

-   -   1. In several studies it was shown that QC catalyses the        formation of pyroGlu residues from glutamate at N-terminus of Aβ        (Cynis et al., Biochim Biophys Acta. 2006 October;        1764(10):1618-25, Schilling et al., FEBS Lett. 2004 Apr. 9;        563(1-3):191-6);    -   2. Both Aβ peptides and QC are expressed in large quantities in        hippocampus and cortex. These brain areas are at particular risk        in AD (Pohl et al., Proc Natl Acad Sci USA. 1991 Nov. 15;        88(22):10059-63, Selkoe, Physiol Rev. 2001 April; 81(2):741-66);    -   3. The APP is cleaved by β-secretase during the transport to the        plasma membrane whereby the N-terminus of Aβ with the free        glutamate residue can be produced (Greenfield et al., Proc Natl        Acad Sci USA. 1999 Jan. 19; 96(2):742-7). In the secretory        vesicles a co-localisation of processed APP and the QC was        determined. So in the mild acid milieu of the vesicles an        accelerated modification of glutamate residue to pyroglutamate        can occur.    -   4. Also other neurodegenerative diseases (familiar Danish (FDD)        or British dementia (FBD)) are related with N-terminal pyroGlu        modified peptides e.g. Bri2, but in contrast they are not        related to Aβ in terms of their primary structure (Vidal R. et        al., 1999 Proc. Natl. Acad. Sci. U.S.A. 97, 4920-4925).

Possibly the QC-catalysed formation of pyroGlu Aβ is involved in thedevelopment and progression of neurodegenerative diseases. The formationof N-terminal modified amyloid peptides certainly represents afundamental factor in the process of Aβ aggregation and could be theonset of disease. The suppression of the pyroGlu Aβ formation byinhibition of QC, might represent a therapeutic approach. QC inhibitorswould be able to prevent the formation of pyroGlu Aβ, reduce theconcentration of pyroglutamate Aβ in the brain and so delay theoligomerisation of Aβ-peptides. Schilling et al. show, that QCexpression was up regulated in the cortex of AD patients and correlatedwith the appearance of pyroGlu-modified Aβ-peptide. Oral application ofa QC inhibitor resulted in reduced pyroglutamate modified AβpE(3-42)level in two different transgenic mouse models of AD and in a newDrosophila model (Schilling et al., 2008 Biol. Chem. (389), 983-991).

Lewy body dementia (LBD) is a neurodegenerative disorder that can occurin persons older than 65 years of age, and typically causes symptoms ofcognitive (thinking) impairment and abnormal behavioral changes.Symptoms can include cognitive impairment, neurological signs, sleepdisorder, and autonomic failure. Cognitive impairment is the presentingfeature of LBD in most cases. Patients have recurrent episodes ofconfusion that progressively worsen. The fluctuation in cognitiveability is often associated with shifting degrees of attention andalertness. Cognitive impairment and fluctuations of thinking may varyover minutes, hours, or days. Lewy bodies are formed from phosphorylatedand nonphosphorylated neurofilament proteins; they contain the synapticprotein alpha-synuclein as well as ubiquitin, which is involved in theelimination of damaged or abnormal proteins. In addition to Lewy Bodies,Lewy neurites, which are inclusion bodies in the cell processes of thenerve cells, may also be present. Amyloid plaques may form in the brainsof patients afflicted with DLB, however they tend to be fewer in numberthan seen in patients with Alzheimer's disease. Neurofibrillary tangles,the other micropathological hallmark of AD, are not a maincharacteristic of LBD but are frequently present in addition to amyloidplaques.

Amyotrophic lateral sclerosis (ALS) is characterized by degeneration ofupper and lower motor neurons. In some ALS patients, dementia or aphasiamay be present (ALS-D). The dementia is most commonly a frontotemporaldementia (FTD), and many of these cases have ubiquitin-positive,tau-negative inclusions in neurons of the dentate gyrus and superficiallayers of the frontal and temporal lobes.

Inclusion-body myositis (IBM) is a crippling disease usually found inpeople over age 50, in which muscle fibers develop inflammation andbegin to atrophy—but in which the brain is spared and patients retaintheir full intellect. Two enzymes involved in the production ofamyloid-β protein were found to be increased inside the muscle cells ofpatients with this most common, progressive muscle disease of olderpeople, in which amyloid-β is also increased.

Another disease that is based on or associated with the accumulation anddeposit of amyloid-like protein is macular degeneration. Maculardegeneration is a common eye disease that causes deterioration of themacula, which is the central area of the retina (the paper-thin tissueat the back of the eye where light-sensitive cells send visual signalsto the brain). Sharp, clear, “straight ahead” vision is processed by themacula. Damage to the macula results in the development of blind spotsand blurred or distorted vision. Age-related macular degeneration (AMD)is a major cause of visual impairment in the United States and forpeople over age 65 it is the leading cause of legal blindness amongCaucasians. Approximately 1.8 million Americans of age 40 and older haveadvanced AMD, and another 7.3 million people with intermediate AMD areat substantial risk for vision loss. The government estimates that by2020 there will be 2.9 million people with advanced AMD. Victims of AMDare often surprised and frustrated to find out how little is known aboutthe causes and treatment of this blinding condition.

There are two forms of macular degeneration: dry macular degenerationand wet macular degeneration. The dry form, in which the cells of themacula slowly begin to break down, is diagnosed in 85 percent of maculardegeneration cases. Both eyes are usually affected by dry AMD, althoughone eye can lose vision while the other eye remains unaffected. Drusen,which are yellow deposits under the retina, are common early signs ofdry AMD. The risk of developing advanced dry AMD or wet AMD increases asthe number or size of the drusen increases. It is possible for dry AMDto advance and cause loss of vision without turning into the wet form ofthe disease; however, it is also possible for early-stage dry AMD tosuddenly change into the wet form.

The wet form, although it only accounts for 15 percent of the cases,results in 90 percent of the blindness, and is considered advanced AMD(there is no early or intermediate stage of wet AMD). Wet AMD is alwayspreceded by the dry form of the disease. As the dry form worsens, somepeople begin to have abnormal blood vessels growing behind the macula.These vessels are very fragile and will leak fluid and blood (hence‘wet’ macular degeneration), causing rapid damage to the macula.

The dry form of AMD will initially often cause slightly blurred vision.The center of vision in particular may then become blurred and thisregion grows larger as the disease progresses. No symptoms may benoticed if only one eye is affected. In wet AMD, straight lines mayappear wavy and central vision loss can occur rapidly.

Diagnosis of macular degeneration typically involves a dilated eye exam,visual acuity test, and a viewing of the back of the eye using aprocedure called fundoscopy to help diagnose AMD, and—if wet AMD issuspected—fluorescein angiography may also be performed. If dry AMDreaches the advanced stages, there is no current treatment to preventvision loss. However, a specific high dose formula of antioxidants andzinc may delay or prevent intermediate AMD from progressing to theadvanced stage. Macugen® (pegaptanib sodium injection), laserphotocoagulation and photodynamic therapy can control the abnormal bloodvessel growth and bleeding in the macula, which is helpful for somepeople who have wet AMD; however, vision that is already lost will notbe restored by these techniques. If vision is already lost, low visionaids exist that can help improve the quality of life.

One of the earliest signs of age-related macular degeneration (AMD) isthe accumulation of extracellular deposits known as drusen between thebasal lamina of the retinal pigmented epithelium (RPE) and Bruch'smembrane (BM). Recent studies conducted by Anderson et al. haveconfirmed that drusen contain amyloid beta. (Experimental Eye Research78 (2004) 243-256).

Pyroglutamated Aβ peptides have been shown to play a key role inaccumulation of Aβ peptides and in plaque formation in Alzheimer'sdiseases. Due to their hydrophobic potential it has been shown thatthese peptides promote aggregation and plaque formation. It has furtherbeen shown in a transgenic mouse model expressing Aβ N3pE-42 in neuronsthat this peptide is neurotoxic in vivo and leads to loss of neurons(Wirths et al. (2009) Acta Neuropatho/118, 487-496).

Antibodies with specificities against the N-terminal pyroglutamate of Aβpeptides are believed to be advantageous because of their specificitytowards only the pathogenic species of Aβ, which carry a pyroglutamateat the N-terminus, but not detecting APP or other Aβ species w/o theN-terminal pyroglutamate. It is thus believed that the risk of potentialside effects, such as uncontrollable cerebral inflammation, will bereduced by use of the antibodies of the invention compared to antibodiesdirected to other Aβ species that the pyroglutamated variants.

Antibodies targeting Aβ N3pE peptides are known (Acero et al (2009) JNeuroimmunol 213, 39-46; Saido et al. (1996) Neuron 14, 457-466; U.S.Pat. No. 7,122,374 and WO 2012/136552).

However, there is a need for humanized antibodies with specificity forAβ N3pE peptides that can be used in human treatment and that positivelyaffect amyloidosis, in particular cognition in diseases and conditionswhere Aβ N3pE may be involved, such as clinical or pre-clinicalAlzheimer's disease, Down's syndrome, and clinical or pre-clinicalcerebral amyloid angiopathy.

SUMMARY OF THE INVENTION

The invention provides novel methods and compositions comprising highlyspecific and highly effective antibodies, including chimeric antibodiesand fragments thereof, including partially or fully humanized antibodiesand fragments thereof, having the ability to specifically recognize andbind to specific epitopes from a range of β-amyloid antigens, inparticular Aβ N3pE peptides, which may be presented to the antibody in amonomeric, dimeric, trimeric, etc, or a polymeric form, in form of anaggregate, fibers, filaments or in the condensed form of a plaque.

In particular, the present invention pertains to a humanized antibody ora functional variant thereof, wherein the variable part of the lightchain of said antibody comprises, consists essentially of or consists ofan amino acid sequence of:

(SEQ ID NO: 7) DVVMTQSPLSLPVTLGQPASISCKSSQSLLX ₁SDGKTYLNWFQQRPGQSP RRLX₂YLVSKLDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCVQGTH FPFTFGGGTKVEIK,whereinX₁ is selected from Y and H; andX₂ is selected from A, I and T;or of an amino acid sequence selected from

(SEQ ID NO: 28) DIQMTQSPSSLSASVGDRVTITCRSSQSLVHSDGNTYLHWYQQKPGKAPKLLIYKVSNRFSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCSQSTHVP PTFGQGTKVEIK; and(SEQ ID NO: 36) DVVMTQSPLSLPVTLGQPASISCKSSQSLLYSNGKTYLNWFQQRPGQSPRRLIYVVSKLDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCVQGTHFP FTFGGGTKVEIK.and/orwherein the variable part of heavy chain of said antibody comprises,consists essentially of or consists of an amino acid sequence of:

(SEQ ID NO: 17) QVQLVQSGAEVKKSGASVKVSCKASGYSFTGX ₃TMNWVRQAPGQGLEWMGLINPX ₄NX ₅VTRYNQKFX ₆GRVTX ₇ X ₈RDTSTTTVX ₉MELTSLTSEDTA X₁₀YYCTREAKREWDETYWGQGTLVTVSS;whereinX₃ is selected from Y and H;X₄ is selected from Y and S;X₅ is selected from G, T, A and E;X₆ is selected from K and Q;X₇ is selected from L and I;X₈ is selected from I and T;X₉ is selected from Y and H; andX₁₀ is selected from V and T;or of an amino acid sequence selected from

(SEQ ID NO: 32) EVQLVESGGGLVQPGGSLRLSCAASGFTFSDYGMAWVRQAPGKGLEWVSFISNLAYSIYYADTVTGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARYDYDNILDYVMDYWGQGTLVTVSS, and (SEQ ID NO: 40)QVQLVESGAEVKKPGASVKVSCKASGYIFNNYWINWVRQAPGQGLEWMGQIYPGDGDTNYNGKFKGRVTITADESTSTAYMELSSLRSEDTAVYYCAREG YIVYWGQGTLVTVSS.

The invention provides humanized antibodies, or fragments thereof, thatpositively affect diseases and conditions of amyloidosis, where Aβ N3pEmay be involved.

In another embodiment, the invention provides humanized antibodies andfragments thereof that bind to Aβ N3pE peptides in the circulation andtissue, in particular in the brain. The humanized antibodies of theinvention are capable of binding free Aβ N3pE peptide molecules or evenbound forms of Aβ N3pE peptides.

Thus, the present invention further provides humanized antibodies thatalter clearance of soluble and bound forms of Aβ N3pE peptides in thecentral nervous system, such as the brain, and the circulation, such asplasma.

In a further embodiment, the invention provides humanized antibodies andfragments thereof, wherein the humanized antibodies specifically bind tothe pyroglutamate carrying N-terminus of Aβ N3pE.

In yet a further embodiment, the present invention also relates to thehost cells transformed with the vectors or incorporating thepolynucleotides that express the humanized antibodies or fragmentsthereof.

Moreover, the present invention provides pharmaceutical compositionscomprising the humanized antibodies of the invention and fragmentsthereof.

The invention further relates to the use of the humanized antibodies andfragments thereof are useful for binding to and clearing or removing ofAβ N3pE in humans and thereby for diagnosing, preventing and treatingdiseases and conditions characterized by amyloidosis or Aβ N3pEtoxicity.

In a particular embodiment, the humanized antibodies of the invention,which are capable of binding to and clearing or removing of Aβ N3pEpeptides in biological fluids and tissues, are useful for the preventionand/or treatment of conditions associated with the formation of AβN3pE-containing plaques, such as diffuse, neuritic, and cerebrovascularplaques in the brain.

The administration of the humanized antibodies of the invention,including immunologically reactive fragments thereof, may lead to theclearance or removal of Aβ N3pE from the aforementioned plaques or otherbiological complexes. Thus, the humanized antibody of the invention willreadily be transport in the circulation, other body fluids and to siteswhere the aforementioned plaques and/or other biological complexes areformed or elsewhere where A□ N3pE exhibits damaging effects.

In addition, removal of Aβ N3pE from plaques or other biologicalcomplexes by the humanized antibodies of the invention may lead to thesolubilization of insoluble forms of plaques and thus lead to theremoval of complete plaques from the affected tissue, such as braintissue. This, in turn, may lead to improvement of cognition in patientsdiagnosed with a neurodegenerative disease, such as mild cognitiveimpairment (MCI), Alzheimer's disease (AD), like for instance sporadicAlzheimer's disease (SAD) or Familial Alzheimer's dementias (FAD) likeFamilial British Dementia (FBD) and Familial Danish Dementia (FDD) orothers, neurodegeneration in Down Syndrome, Lewy body dementia,hereditary cerebral hemorrhage with amyloidosis (Dutch type); the GuamParkinson-Dementia complex.

The binding of the humanized antibodies of the invention to Aβ N3pE inthe circulation or other body fluids may further result to the removalof the circulating or soluble forms of Aβ N3pE. As discussed above, AβN3pE exhibits a high hydrophobicity and has a high affinity to other,e.g. nonpyroglutamated Aβ peptides, which results in the formation ofoligomeric and supermolecular structures, such as amyloid plaques. Ithas been shown that in particular these oligomeric structures are highlyneurotoxic. The formation of oligomeric structures leads to cell damageand death of neuronal cells. Thus, the removal of circulating or solubleforms of Aβ N3pE or even of oligomers comprising Aβ N3pE leads to theprevention of cell damage and/or neurotoxicity. Thus, the invention alsoprovides methods of preventing of neurodegenerative disease, such asmild cognitive impairment (MCI), Alzheimer's disease (AD), like forinstance sporadic Alzheimer's disease (SAD) or Familial Alzheimer'sdementias (FAD) like Familial British Dementia (FBD) and Familial DanishDementia (FDD) or others, neurodegeneration in Down Syndrome, Lewy bodydementia, hereditary cerebral hemorrhage with amyloidosis (Dutch type),the Guam Parkinson-Dementia complex.

The invention further provides methods of preventing and/or treating ofother diseases which are based on or associated with amyloid-likeproteins, in particular Aβ N3pE, such as progressive supranuclear palsy,multiple sclerosis, Creutzfeld Jacob disease, Parkinson's disease,HIV-related dementia, ALS (amyotropic lateral sclerosis), dementiarelated to Adult Onset Diabetes; senile cardiac amyloidosis, and others,including macular degeneration.

The invention further provides a highly sensitive and concomitantlyrobust detection technique that allows quantitative determination of Aβvariants, in particular Aβ N3pE, in biological samples, e.g. liquor orserum samples, preferably serum samples, or tissue samples. This is atremendous challenge, taking the low abundance of these Aβ N3pE peptidesin blood into account. Having such a detection technique available is,however, a prerequisite for studying efficacy of small moleculeinhibitors in drug screening and drug development programs.

The antibodies enabled by the teaching of the present invention areparticularly useful for diagnosis of amyloidosis, a group of diseasesand disorders associated with amyloid plaque formation includingsecondary amyloidosis and age-related amyloidosis including, but notlimited to, neurological disorders such as Alzheimer's Disease (AD),Lewy body dementia, Down's syndrome, hereditary cerebral hemorrhage withamyloidosis (Dutch type), the Guam Parkinson-Dementia complex, as wellas other diseases which are based on or associated with amyloid-likeproteins such as progressive supranuclear palsy, multiple sclerosis;Creutzfeld Jacob disease, hereditary cerebral hemorrhage withamyloidosis Dutch type, Parkinson's disease, HIV-related dementia, ALS(amyotropic lateral sclerosis), dementia related to Adult OnsetDiabetes, senile cardiac amyloidosis, and others, including maculardegeneration, to name just a few.

DESCRIPTION OF THE FIGURES

FIG. 1 shows the CDR definition of mouse antibody clone#6 Amino acidsequence of variable domain of light chain (LC) and heavy chain (HC) areshown. The three CDRs of LC and HC are framed (selected by Sircar etal., 2009 (Sircar A. et al.: RosettaAntibody: antibody variable regionhomology modeling server. Nucleic Acids Research, 2009, Vol. 37, pp.W474-W479) according to Kabat and Wu, 1971 (Kabat E. A. and WU, T. T.:An Attempt to Locate the Non-helical and Permissively Helical Sequencesof Proteins: Application to the Variable Regions of Immunoglobulin Lightand Heavy Chains. Proc. Nat. Acad. Sci. USA; Vol. 68, No. 7, pp.1501-1506, 1971) and Kabat et al. 1991 (Kabat E. A. and WU, T. T.:IDENTICAL V REGION AMINO ACID SEQUENCES AND SEGMENTS OF SEQUENCES INANTIBODIES OF DIFFERENT SPECIFICITIES: Relative Contributions of VH andVL Genes, Minigenes, and Complementarity-Determining Regions to Bindingof Antibody-Combining Sites. The Journal of Immunology, Vol. 147; pp.1709-1719, 1991); beside CDR1 of HC which is defined according toClothia et al., 1989 (Clothia C. et al.: Conformations of immunoglobulinhypervariable regions. Nature, Vol. 342, pp. 877-883, 1989).

FIG. 2 shows the purification of humanized antibody clone#6 by Protein Gchromatography. Recombinant produced humanized antibody clone#6 waspurified by Protein G Chromatography. 24 μl of input fraction (lane 1),flow through fraction (lane 22) and elution fraction (lane 3) are loadedonto 10% SDS PAGE under non reducing conditions. 2 μg of and humanized(lane 4) antibody were compared to mouse antibody (lane 5) in 10%coomassie stained SDS gel.

FIG. 3 shows the calculation of KD of humanized antibody clone#6 HC T97variant and LC L41 variant. The binding affinities of HC T97 and LC L41variant of humanized antibody clone#6 to Aβ(pE3-18) was measured by SPRusing peptide concentrations of 1-100 nM (HC T97 variant) and 10-1000 nM(LC L41 variant). The KD is calculated with 5.3 nM. The KD value of LCL41 variant was determined with 162.7 nM by plotting RU_(equ) valuesagainst the peptide concentration and fitting by steady-state model asdescribed above

FIG. 4 shows the generation of stable cell line expressing humanizedantibody clone #6 variant HC T97. A) MTX Treatment of CHO DG44 cellsstable expressing humanized antibody clone#6 variant HC T97. 0.5 μM MTX(lane 2) leads to increasing expression in comparison with 0.1 μM andwithout MTX (lane 1 and 4). More over low amount of cells dying comparedwith treatment using 1 μM MTX (lane 4). 24 μl supernatant were loadedonto a 10% SDS PAGE under non reducing conditions. B) 18 clones wereyielded after clonal selection by limiting dilution and analyzed byWestern blot. 24 μl of supernatant were loaded onto a 12% SDS PAGE undernon reducing conditions. C) Five of these 18 clones were scaled up andcultivated for 7 days, so expression levels in supernatant could beanalyzed by SPR.

FIG. 5 shows the ITC measurement of humanized antibody clone#6 withAβpE3-18 peptide. Purified humanized antibody clone#6 HC T97 variant wasused for ITC measurement. Top: raw data of ITC measurement. Bottom:Integration of raw data to represent the concentration of added peptideas a function molar ratio peptide/antibody. The values of thethermodynamic parameters are shown left.

FIG. 6 shows the binding of two antibodies, which either comprise thehuman IgG1 Fc wild-type region of SEQ ID NO: 73 (WT) or the K324A mutantvariant thereof (SEQ ID NO: 74), to the Fc gamma receptor CD16A.

FIG. 7 shows the binding of two antibodies, which either comprise thehuman IgG1 Fc wild-type region of SEQ ID NO: 73 (WT) or the K324A mutantvariant thereof (SEQ ID NO: 74), to the Fc gamma receptor CD32A.

FIG. 8 shows the binding of two antibodies, which either comprise thehuman IgG1 Fc wild-type region of SEQ ID NO: 73 (WT) or the K324A mutantvariant thereof (SEQ ID NO: 74), to the Fc gamma receptor CD32B.

FIG. 9 shows the binding of two antibodies, which either comprise thehuman IgG1 Fc wild-type region of SEQ ID NO: 73 (WT) or the K324A mutantvariant thereof (SEQ ID NO: 74), to the Fc gamma receptor CD64.

FIG. 10 shows the binding analysis of two antibodies, which eithercomprised the human IgG1 Fc wild-type region of SEQ ID NO: 73 (WT) orthe K324A mutant variant thereof (SEQ ID NO: 74), to C1q.

DETAILED DESCRIPTION OF THE INVENTION Definitions

The term “antibody” is used in the broadest sense and specificallycovers intact monoclonal antibodies, polyclonal antibodies,multispecific antibodies (e.g. bispecific antibodies) formed from atleast two intact antibodies, and antibody fragments so long as theyexhibit the desired biological activity. The antibody may be an IgM, IgG(e.g. IgG1, IgG2, IgG3 or IgG4), IgD, IgA or IgE, for example.Preferably however, the antibody is not an IgM antibody.

“Antibody fragments” comprise a portion of an intact antibody, generallythe antigen binding or variable region of the intact antibody. Examplesof antibody fragments include Fab, Fab′, F(ab′)2, and Fv fragments:diabodies; single-chain antibody molecules; and multispecific antibodiesformed from antibody fragments.

The term “monoclonal antibody” as used herein refers to an antibodyobtained from a population of substantially homogeneous antibodies, i.e.the individual antibodies comprising the population are identical exceptfor possible naturally occurring mutations that may be present in minoramounts. Monoclonal antibodies are highly specific, being directedagainst a single antigenic site. Furthermore, in contrast to “polyclonalantibody” preparations which typically include different antibodiesdirected against different determinants (epitopes), each monoclonalantibody is directed against a single determinant on the antigen. Inaddition to their specificity, the monoclonal antibodies can frequentlybe advantageous in that they are synthesized by the hybridoma culture,uncontaminated by other immunoglobulins. The “monoclonal” indicates thecharacter of the antibody as being obtained from a substantiallyhomogeneous population of antibodies, and is not to be construed asrequiring production of the antibody by any particular method. Forexample, the monoclonal antibodies to be used in accordance with thepresent invention may be made by the hybridoma method first described byKöhler et al., Nature, 256:495 (1975), or may be made by generally wellknown recombinant DNA methods. The “monoclonal antibodies” may also beisolated from phage antibody libraries using the techniques described inClackson et al., Nature, 352:624-628 (1991) and Marks et al., J. Mol.Biol., 222:581-597 (1991), for example.

The monoclonal antibodies herein specifically include chimericantibodies (immunoglobulins) in which a portion of the heavy and/orlight chain is identical with or homologous to corresponding sequencesin antibodies derived from a particular species or belonging to aparticular antibody class or subclass, while the remainder of thechain(s) is identical with or homologous to corresponding sequences inantibodies derived from another species or belonging to another antibodyclass or subclass, as well as fragments of such antibodies, so long asthey exhibit the desired biological activity.

“Humanized” forms of non-human (e.g., murine) antibodies areimmunoglobulins, immunoglobulin chains or fragments thereof (such as Fv,Fab, Fab′, F(ab′)2 or other antigen-binding subsequences of antibodies)which contain a minimal sequence derived from a non-humanimmunoglobulin. For the most part, humanized antibodies are humanimmunoglobulins (recipient antibody) in which residues from acomplementarity-determining region (CDR) of the recipient are replacedby residues from a CDR of a non-human species (donor antibody) such asmouse, rat or rabbit having the desired specificity, affinity, andcapacity. In some instances, Fv framework region (FR) residues of thehuman immunoglobulin are replaced by corresponding non-human residues.Furthermore, humanized antibodies may comprise residues which are foundneither in the recipient antibody nor in the imported CDR or frameworksequences.

These modifications are made to further refine and optimize antibodyperformance. In general, the humanized antibody will comprisesubstantially all of at least one, and typically two, variable domains,in which all or substantially all of the CDR regions correspond to thoseof a non-human immunoglobulin and all or substantially all of the FRregions are those of a human immunoglobulin sequence. The humanizedantibody optimally also will comprise at least a portion of animmunoglobulin constant region (Fc), typically that of a humanimmunoglobulin. For further details, see Jones et al., Nature,321:522-525 (1986), Reichmann et al, Nature. 332:323-329 (1988): andPresta, Curr. Op. Struct. Biel., 2:593-596 (1992).

The term “therapeutically effective amount” as used herein and in theappended claims means that the amount of humanized antibody administeredis of sufficient quantity to achieve the intended purpose, such as, inthis case, the removal of circulating or soluble forms of pyroglutamatedamyloid beta (Aβ N3pE) peptide and variants thereof.

“Single-chain Fv” or “sFv” antibody fragments comprise the V_(H) andV_(L) domains of antibody, wherein these domains are present in a singlepolypeptide chain. Generally, the Fv polypeptide further comprises apolypeptide linker between the V_(H) and V_(L) domains which enables thesFv to form the desired structure for antigen binding. For a review ofsFv see Plückthun in The Pharmacology of Monoclonal Antibodies, vol.113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315(1994).

The term “diabodies” refers to small antibody fragments with twoantigen-binding sites, which fragments comprise a heavy-chain variabledomain (V_(H)) connected to a light-chain variable domain (V_(D)) in thesame polypeptide chain (V_(H)-V_(D)). By using a linker that is tooshort to allow pairing between the two domains on the same chain, thedomains are forced to pair with the complementary domains of anotherchain and create two antigen-binding sites. Diabodies are described morefully in Hollinger et al., Proc. Natl. Acad. Sol. USA, 90:6444-6448(1993).

An “isolated” antibody is one which has been identified and separatedand/or recovered from a component of its natural environment.Contaminant components of its natural environment are materials whichwould interfere with diagnostic or therapeutic uses for the antibody,and may include enzymes, hormones, and other proteinaceous ornon-proteinaceous solutes. In preferred embodiments, the antibody willbe purified (1) to greater than 95% by weight of antibody as determinedby the Lowry method, and most preferably more than 99% by weight, (2) toa degree sufficient to obtain at least 15 residues of N-terminal orinternal amino acid sequence by use of a spinning cup sequenator, or (3)to homogeneity by SDS-PAGE under reducing or nonreducing conditionsusing Coomassie blue or, preferably, silver stain. Isolated antibodyincludes the antibody in situ within recombinant cells since at leastone component of the antibody's natural environment will not be present.Ordinarily, however, isolated antibody will be prepared by at least onepurification step.

As used herein, the expressions “cell”, “cell line,” and “cell culture”are used interchangeably and all such designations include progeny.Thus, the words “transformants” and “transformed cells” include theprimary subject cell and culture derived therefrom without regard forthe number of transfers. It is also understood that all progeny may notbe precisely identical in DNA content, due to deliberate or inadvertentmutations. Mutant progeny that have the same function or biologicalactivity as screened for in the originally transformed cell areincluded. Where distinct designations are intended, this will be clearfrom the context.

The terms “polypeptide”, “peptide”, and “protein”, as used herein, areinterchangeable and are defined to mean a biomolecule composed of aminoacids linked by a peptide bond.

If peptide or amino acid sequences are mentioned herein, each amino acidresidue is represented by a one-letter or a three-letter designation,corresponding to the trivial name of the amino acid, in accordance withthe following conventional list:

Amino Acid One-Letter Symbol Three-Letter Symbol Alanine A Ala ArginineR Arg Asparagine N Asn Aspartic acid D Asp Cysteine C Cys Glutamine QGln Glutamic acid E Glu Glycine G Gly Histidine H His Isoleucine I IleLeucine L Leu Lysine K Lys Methionine M Met Phenylalanine F Phe ProlineP Pro Serine S Ser Threonine T Thr Tryptophan W Trp Tyrosine Y TyrValine V Val

The terms “a”, “an” and “the” as used herein are defined to mean “one ormore” and include the plural unless the context is inappropriate.

The language “diseases and disorders which are caused by or associatedwith amyloid or amyloid-like proteins” includes, but is not limited to,diseases and disorders caused by the presence or activity ofamyloid-like proteins in monomeric, fibril, or polymeric state, or anycombination of the three. Such diseases and disorders include, but arenot limited to, amyloidosis, endocrine tumors, and macular degeneration.

The term “amyloidosis” refers to a group of diseases and disordersassociated with amyloid plaque formation including, but not limited to,secondary amyloidosis and age-related amyloidosis such as diseasesincluding, but not limited to, neurological disorders such asAlzheimer's Disease (AD), including diseases or conditions characterizedby a loss of cognitive memory capacity such as, for example, mildcognitive impairment (MCI), sporadic Alzheimer's disease, Lewy bodydementia, Down's syndrome, hereditary cerebral hemorrhage withamyloidosis (Dutch type); the Guam Parkinson-Dementia complex, familialforms of Alzheimer's disease like Familial British Dementia (FBD) andFamilial Danish Dementia (FDD); as well as other diseases which arebased on or associated with amyloid-like proteins such as progressivesupranuclear palsy, multiple sclerosis; Creutzfeld Jacob disease,Parkinson's disease, HIV-related dementia, ALS (amyotropic lateralsclerosis), inclusion-body myositis (IBM), Adult Onset Diabetes, andsenile cardiac amyloidosis; and various eye diseases including maculardegeneration, drusen-related optic neuropathy, and cataract due tobeta-amyloid deposition.

“Amyloid β, Aβ or /β-amyloid” is an art recognized term and refers toamyloid p proteins and peptides, amyloid β precursor protein (APP), aswell as modifications, fragments and any functional equivalents thereof.In particular, by amyloid β as used herein is meant any fragmentproduced by proteolytic cleavage of APP but especially those fragmentswhich are involved in or associated with the amyloid pathologiesincluding, but not limited to, Aβ₁₋₃₈, Aβ₁₋₄₀, Aβ₁₋₄₂. The amino acidsequences of these Aβ peptides are as follows:

Aβ 1-42 (SEQ ID NO. 1): Asp-Ala-Glu-Phe-Arg-His-Asp-Ser-Gly-Tyr-Glu-Val-His-His-Gln-Lys-Leu-Val-Phe-Phe-Ala-Glu-Asp-Val-Gly-Ser-Asn-Lys-Gly-Ala-Ile-Ile-Gly-Leu-Met-Val- Gly-Gly-Val-Val-Ile-AlaAβ 1-40 (SEQ ID NO. 2): Asp-Ala-Glu-Phe-Arg-His-Asp-Ser-Gly-Tyr-Glu-Val-His-His-Gln-Lys-Leu-Val-Phe-Phe-Ala-Glu-Asp-Val-Gly-Ser-Asn-Lys-Gly-Ala-Ile-Ile-Gly-Leu-Met-Val- Gly-Gly-Val-Val Aβ 1-38(SEQ ID NO. 3): Asp-Ala-Glu-Phe-Arg-His-Asp-Ser-Gly-Tyr-Glu-Val-His-His-Gln-Lys-Leu-Val-Phe-Phe-Ala-Glu-Asp-Val-Gly-Ser-Asn-Lys-Gly-Ala-Ile-Ile-Gly-Leu-Met-Val- Gly-Gly

“pGlu-Aβ” or “Aβ N3pE” refers to N-terminally truncated forms of Aβ,that start at the glutamic acid residue at position 3 in the amino acidsequence of Aβ, and wherein said glutamic acid residue is cyclized toform a pyroglutamic acid residue. In particular, by pGlu-Aβ or Aβ N3pEas used herein are meant those fragments which are involved in orassociated with the amyloid pathologies including, but not limited to,pGlu-Aβ₃₋₃₈, pGlu-Aβ₃₋₄₀, p-Glu-Aβ₃₋₄₂.

The sequences of the N-terminally truncated forms of Aβ, Aβ₃₋₃₈, Aβ₃₋₄₀,Aβ₃₋₄₂ are as follows:

Aβ 3-42 (SEQ ID NO. 4): Glu-Phe-Arg-His-Asp-Ser-Gly-Tyr-Glu-Val-His-His-Gln-Lys-Leu-Val-Phe-Phe-Ala-Glu-Asp-Val-Gly-Ser-Asn-Lys-Gly-Ala-Ile-Ile-Gly-Leu-Met-Val-Gly-Gly- Val-Val-Ile-Ala Aβ 3-40(SEQ ID NO. 5): Glu-Phe-Arg-His-Asp-Ser-Gly-Tyr-Glu-Val-His-His-Gln-Lys-Leu-Val-Phe-Phe-Ala-Glu-Asp-Val-Gly-Ser-Asn-Lys-Gly-Ala-Ile-Ile-Gly-Leu-Met-Val-Gly-Gly- Val-Val Aβ 3-38 (SEQ IDNO. 6): Glu-Phe-Arg-His-Asp-Ser-Gly-Tyr-Glu-Val-His-His-Gln-Lys-Leu-Val-Phe-Phe-Ala-Glu-Asp-Val-Gly-Ser-Asn-Lys-Gly-Ala-Ile-Ile-Gly-Leu-Met-Val-Gly-Gly

The present invention pertains to humanized antibodies specific forhuman Aβ peptides that are N-terminally truncated by cleaving off orloosing amino acids no. 1 and 2 of the N-terminus and in which the souncovered N-terminal amino acid no. 3 is modified by pyroglutamateformation and which thus bear a pyroglutamate residue at position 3 ofthe N-terminus (further referred to as Aβ N3pE peptides or N3pE-Aβpeptides or pyroglutamated Aβ peptides).

In a first aspect, the present invention pertains to a humanizedantibody, wherein the variable part of the light chain of said antibodycomprises, consists essentially of or consists of an amino acid sequenceof:

(SEQ ID NO: 7) DVVMTQSPLSLPVTLGQPASISCKSSQSLLX ₁SDGKTYLNWFQQRPGQSP RRLX₂YLVSKLDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCVQGTH FPFTFGGGTKVEIK,whereinX₁ is selected from Y and H; andX₂ is selected from A, I and T.

In a preferred embodiment of the present invention, the antibody havingthe variable part of the light chain of said antibody, which comprises,consists essentially of or consists of the amino acid sequence of SEQ IDNO: 7, comprises the following CDR regions in the light chain:

V_(L) CDR1: (SEQ ID NO: 8) KSSQSLLX ₁SDGKTYLN, wherein X₁ is selectedfrom Y and H; V_(L) CDR2: (SEQ ID NO: 9) LVSKLDS; and V_(L) CDR3: (SEQID NO: 10) VQGTHFP.

More preferably, in the variable part of the light chain of the antibodyof the present invention, X₁ is Y and X₂ is I, and the variable part ofthe light chain thus comprises, consists essentially of or consists ofthe amino acid sequence of:

(SEQ ID NO: 11) DVVMTQSPLSLPVTLGQPASISCKSSQSLLYSDGKTYLNWFQQRPGQSPRRLIYLVSKLDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCVQGTHFP FTFGGGTKVEIK.

In a preferred embodiment of the present invention, the antibody havingthe variable part of the light chain of said antibody, which comprises,consists essentially of or consists of the amino acid sequence of SEQ IDNO: 11, comprises the following CDR regions in the light chain:

V_(L) CDR1: (SEQ ID NO: 12) KSSQSLLYSDGKTYLN, V_(L) CDR2: (SEQ ID NO: 9)LVSKLDS; and V_(L) CDR3: (SEQ ID NO: 10) VQGTHFP.

Even preferably, in the variable part of the light chain of the antibodyof the present invention, X₁ is Y and X₂ is A and the variable part ofthe light chain thus comprises, consists essentially of or consists ofthe amino acid sequence of:

(SEQ ID NO: 13) DVVMTQSPLSLPVTLGQPASISCKSSQSLLYSDGKTYLNWFQQRPGQSPRRLAYLVSKLDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCVQGTHFP FTFGGGTKVEIK.

In a preferred embodiment of the present invention, the antibody havingthe variable part of the light chain of said antibody, which comprises,consists essentially of or consists of the amino acid sequence of SEQ IDNO: 13, comprises the CDR regions V_(L) CDR1 of SEQ ID NO: 12, V_(L)CDR2 SEQ ID NO: 9 and V_(L) CDR3 of SEQ ID NO: 10.

Most preferably, in the variable part of the light chain of the antibodyof the present invention, X₁ is Y and X₂ is T and the variable part ofthe light chain thus comprises, consists essentially of or consists ofthe amino acid sequence of:

(SEQ ID NO: 14) DVVMTQSPLSLPVTLGQPASISCKSSQSLLYSDGKTYLNWFQQRPGQSPRRLTYLVSKLDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCVQGTHFP FTFGGGTKVEIK.

In a preferred embodiment of the present invention, the antibody havingthe variable part of the light chain of said antibody, which comprises,consists essentially of or consists of the amino acid sequence of SEQ IDNO: 14, comprises the CDR regions V_(L) CDR1 of SEQ ID NO: 12, V_(L)CDR2 SEQ ID NO: 9 and V_(L) CDR3 of SEQ ID NO: 10.

Even most preferably, in the variable part of the light chain of theantibody of the present invention, X₁ is H and X₂ is T and the lightchain thus comprises, consists essentially of or consists of the aminoacid sequence of:

(SEQ ID NO: 15) DVVMTQSPLSLPVTLGQPASISCKSSQSLLHSDGKTYLNWFQQRPGQSPRRLTYLVSKLDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCVQGTHFP FTFGGGTKVEIK.

In a preferred embodiment of the present invention, the antibody havingthe variable part of the light chain of said antibody, which comprises,consists essentially of or consists of the amino acid sequence of SEQ IDNO: 15, comprises the following CDR regions in the light chain:

V_(L) CDR1: (SEQ ID NO: 16) KSSQSLLHSDGKTYLN, V_(L) CDR2: (SEQ ID NO: 9)LVSKLDS; and V_(L) CDR3: (SEQ ID NO: 10) VQGTHFP.

Further in accordance with the first aspect, present invention pertainsto a humanized antibody, wherein the variable part of heavy chain ofsaid antibody comprises, consists essentially of or consists of an aminoacid sequence of:

(SEQ ID NO: 17) QVQLVQSGAEVKKSGASVKVSCKASGYSFTGX ₃TMNWVRQAPGQGLEWMGLINPX ₄NX ₅VTRYNQKFX ₆GRVTX ₇ X ₈RDTSTTTVX ₉MELTSLTSEDTA X₁₀YYCTREAKREWDETYWGQGTLVTVSS;whereinX₃ is selected from Y and H;X₄ is selected from Y and S;X₅ is selected from G, T, A and E;X₆ is selected from K and Q;X₇ is selected from L and I;X₈ is selected from I and T;X₉ is selected from Y and H; andX₁₀ is selected from V and T;

In a preferred embodiment of the present invention, the antibody havingthe variable part of the heavy chain of said antibody, which comprises,consists essentially of or consists of the amino acid sequence of SEQ IDNO: 17, comprises the following CDR regions in the heavy chain:

V_(H) CDR1: (SEQ ID NO: 18) GYSFTGX ₃TMN, wherein X₃ is selected from Yand H; V_(H) CDR2: (SEQ ID NO: 19) LINPX ₄NX ₅VTRYNQKFX ₆G; wherein X₄is selected from Y and S, X₅ is selected from G, T, A and E and X₆ isselected from K and Q; and V_(H) CDR3: (SEQ ID NO: 20) EAKREWDETY.

Preferably, in the variable part of the heavy chain of the antibody ofthe present invention, X₃ is Y, X₄ is Y, X₅ is G, X₆ is K, X₇ is T, X₈is I, X₉ is Y and X₁₀ is V and the heavy chain thus comprises, consistsessentially of or consists of the amino acid sequence of:

(SEQ ID NO: 21) QVQLVQSGAEVKKSGASVKVSCKASGYSFTGYTMNWVRQAPGQGLEWMGLINPYNGVTRYNQKFKGRVTLIRDTSTTTVYMELTSLTSEDTAVYYCTREA KREWDETYWGQGTLVTVSS.

In a preferred embodiment of the present invention, the antibody havingthe variable part of the heavy chain of said antibody, which comprises,consists essentially of or consists of the amino acid sequence of SEQ IDNO: 21, comprises the following CDR regions in the heavy chain:

V_(H) CDR1: (SEQ ID NO: 22) GYSFTGYTMN, V_(H) CDR2: (SEQ ID NO: 23)LINPYNGVTRYNQKFKG; V_(H) CDR3: (SEQ ID NO: 20) EAKREWDETY.

More preferably, in the variable part of the heavy chain of the antibodyof the present invention, X₃ is H, X₄ is S, X₅ is G, X₆ is Q, X₇ is I,X₈ is T, X₉ is H and X₁₀ is V and the heavy chain thus comprises,consists essentially of or consists of the amino acid sequence of:

(SEQ ID NO: 24) QVQLVQSGAEVKKSGASVKVSCKASGYSFTGHTMNWVRQAPGQGLEWMGLINPSNGVTRYNQKFQGRVTITRDTSTTTVHMELTSLTSEDTAVYYCTREA KREWDETYWGQGTLVTVSS.

In a preferred embodiment of the present invention, the antibody havingthe variable part of the heavy chain of said antibody, which comprises,consists essentially of or consists of the amino acid sequence of SEQ IDNO: 24, comprises the following CDR regions in the heavy chain:

V_(H) CDR1: (SEQ ID NO: 25) GYSFTGHTMN, V_(H) CDR2: (SEQ ID NO: 26)LINPSNGVTRYNQKFQG; V_(H) CDR3: (SEQ ID NO: 20) EAKREWDETY.

Even more preferably, in the variable part of the heavy chain of theantibody of the present invention, X₃ is H, X₄ is S, X₅ is G, X₆ is Q,X₇ is I, X₈ is T, X₉ is H and X₁₀ is T and the heavy chain thuscomprises, consists essentially of or consists of the amino acidsequence of:

(SEQ ID NO: 27) QVQLVQSGAEVKKSGASVKVSCKASGYSFTGHTMNWVRQAPGQGLEWMGLINPSNGVTRYNQKFQGRVTITRDTSTTTVHMELTSLTSEDTATYYCTREA KREWDETYWGQGTLVTVSS.

In a preferred embodiment of the present invention, the antibody havingthe variable part of the heavy chain of said antibody, which comprises,consists essentially of or consists of the amino acid sequence of SEQ IDNO: 27, comprises the following CDR regions in the heavy chain:

V_(H) CDR1: (SEQ ID NO: 25) GYSFTGHTMN, V_(H) CDR2: (SEQ ID NO: 26)LINPSNGVTRYNQKFQG; V_(H) CDR3: (SEQ ID NO: 20) EAKREWDETY.

Most preferably, in the variable part of the heavy chain of the antibodyof the present invention, X₃ is H, X₄ is S, X₅ is T, X₆ is Q, X₇ is I,X₈ is T, X₉ is H and X₁₀ is T and the heavy chain thus comprises,consists essentially of or consists of the amino acid sequence of:

(SEQ ID NO: 66) QVQLVQSGAEVKKSGASVKVSCKASGYSFTGHTMNWVRQAPGQGLEWMGLINPSNTVTRYNQKFQGRVTITRDTSTTTVHMELTSLTSEDTATYYCTREA KREWDETYWGQGTLVTVSS.

In a preferred embodiment of the present invention, the antibody havingthe variable part of the heavy chain of said antibody, which comprises,consists essentially of or consists of the amino acid sequence of SEQ IDNO: 66, comprises the following CDR regions in the heavy chain:

V_(H) CDR1: (SEQ ID NO: 25) GYSFTGHTMN, V_(H) CDR2: (SEQ ID NO: 67)LINPSNTVTRYNQKFQG; V_(H) CDR3: (SEQ ID NO: 20) EAKREWDETY.

Even most preferably, in the variable part of the heavy chain of theantibody of the present invention, X₃ is H, X₄ is S, X₅ is A, X₆ is Q,X₇ is I, X₈ is T, X₉ is H and X₁₀ is T and the heavy chain thuscomprises, consists essentially of or consists of the amino acidsequence of:

(SEQ ID NO: 68) QVQLVQSGAEVKKSGASVKVSCKASGYSFTGHTMNWVNRQAPGQGLEWMGLINPSNAVTRYNQKFQGRVTITRDTSTTTVHMELTSLTSEDTATYYCTRE AKREWDETYWGQGTLVTVSS.

In a preferred embodiment of the present invention, the antibody havingthe variable part of the heavy chain of said antibody, which comprises,consists essentially of or consists of the amino acid sequence of SEQ IDNO: 68, comprises the following CDR regions in the heavy chain:

V_(H) CDR1: (SEQ ID NO: 25) GYSFTGHTMN, V_(H) CDR2: (SEQ ID NO: 69)LINPSNAVTRYNQKFQG; V_(H) CDR3: (SEQ ID NO: 20) EAKREWDETY.

Even most preferably, in the variable part of the heavy chain of theantibody of the present invention, X₃ is H, X₄ is S, X₅ is E, X₆ is Q,X₇ is I, X₈ is T, X₉ is H and X₁₀ is T and the heavy chain thuscomprises, consists essentially of or consists of the amino acidsequence of:

(SEQ ID NO: 70) QVQLVQSGAEVKKSGASVKVSCKASGYSFTGHTMNWVRQAPGQGLEWMGLINPSNEVTRYNQKFQGRVTITRDTSTTTVHMELTSLTSEDTATYYCTREA KREWDETYWGQGTLVTVSS.

In a preferred embodiment of the present invention, the antibody havingthe variable part of the heavy chain of said antibody, which comprises,consists essentially of or consists of the amino acid sequence of SEQ IDNO: 70, comprises the following CDR regions in the heavy chain:

V_(H) CDR1: (SEQ ID NO: 25) GYSFTGHTMN, V_(H) CDR2: (SEQ ID NO: 71)LINPSNEVTRYNQKFQG; V_(H) CDR3: (SEQ ID NO: 20) EAKREWDETY.

Further according to first aspect of the invention, the humanizedantibodies comprising, essentially consisting of or consisting of thefollowing combinations of the variable parts of the light chain andheavy chain are preferred:

Light chain Heavy chain Humanized antibody variable part, variable part,variant SEQ ID NO: SEQ ID NO: General 7 17 i) 11 21 ii) 11 24 iii) 11 27iv) 11 66 v) 11 68 vi) 11 70 vii) 13 21 viii) 13 24 ix) 13 27 x) 13 66xi) 13 68 xii) 13 70 xiii) 14 21 xiv) 14 24 xv) 14 27 Xvi 14 66 Xvii 1468 Xviii 14 70 xix) 15 21 xx) 15 24 xxi) 15 27 Xxii 15 66 Xxiii 15 68Xiv 15 70

More preferably, the variable part of the light chain of the humanizedantibody according to the invention comprises, consists essentially ofor consists of the amino acid sequence of SEQ ID NO: 14.

Even more preferably, the variable part of the heavy chain of thehumanized antibody according to the invention comprises, consistsessentially of or consists of the amino acid sequence of SEQ ID NO: 27.

Even more preferably, the variable part of the heavy chain of thehumanized antibody according to the invention comprises, consistsessentially of or consists of the amino acid sequence selected from SEQID NO: 66, 72 and 74.

Most preferably, the variable part of the heavy chain of the humanizedantibody according to the invention comprises, consists essentially ofor consists of the amino acid sequence of SEQ ID NO: 70.

Most preferably, the variable part of the light chain of the humanizedantibody according to the invention comprises, consists essentially ofor consists of the amino acid sequence of SEQ ID NO: 14 and the variablepart of the heavy chain of the humanized antibody according to theinvention comprises, consists essentially of or consists of the aminoacid sequence of SEQ ID NO: 27.

Even most preferably,

-   -   the variable part of the light chain of the humanized antibody        according to the invention comprises, consists essentially of or        consists of the amino acid sequence of SEQ ID NO: 14; and    -   the variable part of the heavy chain of the humanized antibody        according to the invention comprises, consists essentially of or        consists of the amino acid sequence of SEQ ID NO: 27; and    -   the variable part of the light chain of said antibody, which        comprises, consists essentially of or consists of the amino acid        sequence of SEQ ID NO: 14, comprises the CDR regions V_(L) CDR1        of SEQ ID NO: 12, V_(L) CDR2 SEQ ID NO: 9 and V_(L) CDR3 of SEQ        ID NO: 10; and    -   the variable part of the heavy chain of said antibody, which        comprises, consists essentially of or consists of the amino acid        sequence of SEQ ID NO: 27, comprises the CDR regions V_(H) CDR1        of SEQ ID NO: 25, V_(H) CDR2 SEQ ID NO: 26 and V_(H) CDR3 of SEQ        ID NO: 20.

Even most preferably, the variable part of the light chain of thehumanized antibody according to the invention comprises, consistsessentially of or consists of the amino acid sequence of SEQ ID NO: 14and the variable part of the heavy chain of the humanized antibodyaccording to the invention comprises, consists essentially of orconsists of the amino acid sequence of SEQ ID NO: 70.

Even most preferably,

-   -   the variable part of the light chain of the humanized antibody        according to the invention comprises, consists essentially of or        consists of the amino acid sequence of SEQ ID NO: 14; and    -   the variable part of the heavy chain of the humanized antibody        according to the invention comprises, consists essentially of or        consists of the amino acid sequence of SEQ ID NO: 70; and    -   the variable part of the light chain of said antibody, which        comprises, consists essentially of or consists of the amino acid        sequence of SEQ ID NO: 14, comprises the CDR regions V_(L) CDR1        of SEQ ID NO: 12, V_(L) CDR2 SEQ ID NO: 9 and V_(L) CDR3 of SEQ        ID NO: 10; and    -   the variable part of the heavy chain of said antibody, which        comprises, consists essentially of or consists of the amino acid        sequence of SEQ ID NO: 70, comprises the CDR regions V_(H) CDR1        of SEQ ID NO: 25, V_(H) CDR2 SEQ ID NO: 71 and V_(H) CDR3 of SEQ        ID NO: 20.

In a second aspect, the present invention pertains to a humanizedantibody, wherein the variable part of the light chain of said antibodycomprises, consists essentially of or consists of an amino acid sequenceof:

(SEQ ID NO: 28) DIQMTQSPSSLSASVGDRVTITCRSSQSLVHSDGNTYLHWYQQKPGKAPKLLIYKVSNRFSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCSQSTHVP PTFGQGTKVEIK.

In a preferred embodiment of the present invention, the antibody havingthe variable part of the light chain of said antibody, which comprises,consists essentially of or consists of the amino acid sequence of SEQ IDNO: 28, comprises the following CDR regions in the light chain:

V_(L) CDR1: (SEQ ID NO: 29) RSSQSLVHSDGNTYLH, V_(L) CDR2: (SEQ ID NO:30) KVSNRFS; and V_(L) CDR3: (SEQ ID NO: 31) SQSTHVPPT.

Further in accordance with the second aspect, the present inventionpertains to a humanized antibody, wherein the variable part of the heavychain of said antibody comprises, consists essentially of or consists ofan amino acid sequence of:

(SEQ ID NO: 32) EVQLVESGGGLVQPGGSLRLSCAASGFTFSDYGMAWVRQAPGKGLEWVSFISNLAYSIYYADTVTGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARYDYDNILDYVMDYWGQGTLVTVSS.

In a preferred embodiment of the present invention, the antibody havingthe variable part of the heavy chain of said antibody, which comprises,consists essentially of or consists of the amino acid sequence of SEQ IDNO: 32, comprises the following CDR regions in the heavy chain:

V_(H) CDR1: (SEQ ID NO: 33) GFTFSDYGMA, V_(H) CDR2: (SEQ ID NO: 34)FISNLAYSIYYADTVTG; V_(H) CDR3: (SEQ ID NO: 35) YDYDNILDYVMDY.

In a third aspect, the present invention pertains to a humanizedantibody, wherein the variable part of the light chain of said antibodycomprises, consists essentially of or consists of an amino acid sequenceof:

(SEQ ID NO: 36) DVVMTQSPLSLPVTLGQPASISCKSSQSLLYSNGKTYLNWFQQRPGQSPRRLIYVVSKLDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCVQGTHFP FTFGGGTKVEIK.

In a preferred embodiment of the present invention, the antibody havingthe variable part of the light chain of said antibody, which comprises,consists essentially of or consists of the amino acid sequence of SEQ IDNO: 36, comprises the following CDR regions in the light chain:

V_(L) CDR1: (SEQ ID NO: 37) KSSQSLLYSNGKTYLN, V_(L) CDR2: (SEQ ID NO:38) VVSKLDS; and V_(L) CDR3: (SEQ ID NO: 39) VQGTHFPFT.

Further in accordance with the third aspect, the present inventionpertains to a humanized antibody, wherein the variable part of the heavychain of said antibody comprises, consists essentially of or consists ofan amino acid sequence of:

(SEQ ID NO: 40) QVQLVESGAEVKKPGASVKVSCKASGYIFNNYWINWVRQAPGQGLEWMGQIYPGDGDTNYNGKFKGRVTITADESTSTAYMELSSLRSEDTAVYYCAREG YIVYWGQGTLVTVSS.

In a preferred embodiment of the present invention, the antibody havingthe variable part of the heavy chain of said antibody, which comprises,consists essentially of or consists of the amino acid sequence of SEQ IDNO: 40, comprises the following CDR regions in the heavy chain:

V_(H) CDR1: (SEQ ID NO: 41) GYIFNNY, V_(H) CDR2: (SEQ ID NO: 42)QIYPGDGDTNYNGKFKG; V_(H) CDR3: (SEQ ID NO: 43) EGYIVY.

In a further particularly preferred embodiment, the present inventionpertains to humanized antibodies specific for human N3pE-Aβ peptides,which comprise CDR regions in the light chain selected from:

V_(L) CDR 1 V_(L) CDR 2 V_(L) CDR 3 KSSQSLLHSDGKTYLN LVSKLDS VQGTHFP(SEQ ID NO: 16) (SEQ ID NO: 9) (SEQ ID NO: 10) RSSQSLVHSDGNTYLH KVSNRFSSQSTHVPPT (SEQ ID NO: 29) (SEQ ID NO: 30) (SEQ ID NO: 31)KSSQSLLYSNGKTYLN VVSKLDS VQGTHFPFT (SEQ ID NO: 37) (SEQ ID NO: 38) (SEQID NO: 39)

Furthermore, the present invention pertains to human antibodies specificfor human N3pE-Aβ peptides, which comprise CDR regions in the heavychain selected from:

V_(H) CDR 1 V_(H) CDR 2 V_(H) CDR 3 GYSFTGYTMN LINPYNGVTRYNQKFKGEAKREWDETY (SEQ ID NO: 22) (SEQ ID NO: 23) (SEQ ID NO: 20) GYSFTGHTMNLINPSNGVTRYNQKFQG YDYDNILDYVMDY (SEQ ID NO: 25) (SEQ ID NO: 26) (SEQ IDNO: 35) GFTFSDYGMA FISNLAYSIYYADTVTG EGYIVY (SEQ ID NO: 33) (SEQ ID NO:34) (SEQ ID NO: 43) GYIFNNY QIYPGDGDTNYNGKFKG (SEQ ID NO: 41) (SEQ IDNO: 42) LINPSNTVTRYNQKFQG (SEQ ID NO: 67) LINPSNAVTRYNQKFQG (SEQ ID NO:69) LINPSNEVTRYNQKFQG (SEQ ID NO: 71)

Preferred humanized antibodies according to the invention are humanizedforms of monoclonal mouse antibodies that are produced by a hybridomacell line selected from:

-   -   Aβ 5-5-6 (Deposit No. DSM ACC 2923)    -   Aβ 6-1-6 (Deposit No. DSM ACC 2924)    -   Aβ 17-4-3 (Deposit No. DSM ACC 2925)    -   Aβ 24-2-3 (Deposit No. DSM ACC 2926)        which are described in WO 2010/009987.

The sequences of the light and heavy chains for the humanized antibodiesof the present invention can vary. The immunoglobulins can have twopairs of light chain/heavy chain complexes, at least one chaincomprising one or more mouse complementarity determining regions (CDRs)functionally joined to human framework region segments.

In another embodiment, the present invention is directed to recombinantpolynucleotides encoding the humanized antibodies of the inventioncomprising the heavy and light chain CDRs as set forth herein.

The human framework region of the antibodies of the invention isdetermined by comparison of a framework or variable region amino acidsequence of a CDR-providing non-human immunoglobulin with correspondingsequences in a sequence collection comprising human immunoglobulinvariable regions. A sequence having a high percentage of identical aminoacids is selected.

Preferred polynucleotides of the present invention encode antibodies,comprising CDRs selected from those consisting of the amino acidsequences of SEQ ID NOs: 9, 10, 16, 29-31 and 37-39 in the light chainand selected from those consisting of the amino acid sequences of SEQ IDNOs: 20, 22, 23, 25, 26, 33-35, 41-43, 67, 69 and 71 in the heavy chain.

Further preferred are polynucleotides, which encode antibodies, whereinthe variable part of the light chain comprises, essentially consists orconsists of an amino acid sequence selected from SEQ ID NOs: 7, 11, 13,14, and 28.

Even preferred are polynucleotides, which encode antibodies, wherein thevariable part of the heavy chain comprises, essentially consists orconsists of an amino acid sequence selected from SEQ ID NOs: 17, 21, 24,27, 32, 36, 40, 66, 68 and 70.

In a further embodiment, the humanized antibodies of the presentinvention have a human IgG1 Fc region, which comprises, consistsessentially of or consists of an amino acid sequence of SEQ ID NO: 73.

C1q and two serine proteases, C1r and C1s, form the complex C1, thefirst component of the complement dependent cytotoxicity (CDC) pathway.C1q is a hexavalent molecule with a molecular weight of approximately460,000 and a structure likened to a bouquet of tulips in which sixcollagenous “stalks” are connected to six globular head regions (Burtonand Woof, Advances in Immunol 51:1-84; 1992). Binding of IgG1 moleculesto C1q initiates complement activation and subsequently leads tocomplement-mediated cell lysis. The humanized antibodies of the presentinvention shall be used in treatment of inflammatory diseases andconditions, i.e. the humanized antibodies of the present invention shallhave anti-inflammatory properties.

Effector functions of the humanized antibodies of the invention can alsobe mediated by the interaction of the Fc region of an antibody with Fcreceptors (FcRs), which are specialized cell surface receptors onhematopoietic cells. Fc receptors belong to the immunoglobulinsuperfamily, and have been shown to mediate both the removal ofantibody-coated pathogens by phagocytosis of immune complexes, and thelysing of erythrocytes and various other cellular targets (e.g. tumorcells) coated with the corresponding antibody, via antibody dependentcell mediated cytotoxicity (ADCC) (Van de Winkel and Anderson, J. Leuk.Bioi. 49:511-24; 1991).

Therefore, the present invention further provides humanized antibodiesthat still bind to the Fc receptors to fulfill their effector functions.But, preferably the humanized antibodies of the invention do not show acomplement dependent cytotoxicity. More preferably, the humanizedantibodies of the invention do not activate the complement system, butrather inhibit the complement-mediated cell lysis.

Thus, in a preferred embodiment, the humanized antibodies of the presentinvention have a human IgG Fc region, which comprises one or more anamino acid substitutions, preferably the substitution of 3 or 2 aminoacids, most preferably the substitution of one amino acid. The aminoacid substitutions can be achieved by conventional methods, such assite-directed mutagenesis of the human IgG1 Fc region of the antibodiesof the present invention.

In a more preferred embodiment, the humanized antibodies of the presentinvention have a human IgG Fc region which comprises an amino acidsubstitution at position 324 as shown in SEQ ID NO: 74 [position 324corresponds to position 322 according to EU numbering scheme, Kabat etal., Sequences of Proteins of Immunological Interest, 5th Ed., USDepartment of Health and Human Services, NIH Publication No. 91-3242,National Institutes of Health, Bethesda, Md. (1991); Edelman et al.,PNAS USA 63:78-85 (1969)]. The amino acid substitution is preferablyK324A.

In a most preferred embodiment, the humanized antibodies of the presentinvention have a human IgG Fc region, which comprises, consistsessentially of or consists of an amino acid sequence of SEQ ID NO: 74.

Further most preferably, the variable part of the light chain of thehumanized antibody according to the invention comprises, consistsessentially of or consists of the amino acid sequence of SEQ ID NO: 14;and the variable part of the heavy chain of the humanized antibodyaccording to the invention comprises, consists essentially of orconsists of the amino acid sequence of SEQ ID NO: 27; and the human IgGFc region comprises, consists essentially of or consists of the aminoacid sequence of SEQ ID NO: 74.

Even most preferably,

-   -   the variable part of the light chain of the humanized antibody        according to the invention comprises, consists essentially of or        consists of the amino acid sequence of SEQ ID NO: 14; and    -   the variable part of the heavy chain of the humanized antibody        according to the invention comprises, consists essentially of or        consists of the amino acid sequence of SEQ ID NO: 27; and    -   the human IgG Fc region comprises, consists essentially of or        consists of the amino acid sequence of SEQ ID NO: 74;    -   the variable part of the light chain of said antibody, which        comprises, consists essentially of or consists of the amino acid        sequence of SEQ ID NO: 14, comprises the CDR regions V_(L) CDR1        of SEQ ID NO: 12, V_(L) CDR2 SEQ ID NO: 9 and V_(L) CDR3 of SEQ        ID NO: 10; and    -   the variable part of the heavy chain of said antibody, which        comprises, consists essentially of or consists of the amino acid        sequence of SEQ ID NO: 27, comprises the CDR regions V_(H) CDR1        of SEQ ID NO: 25, V_(H) CDR2 SEQ ID NO: 26 and V_(H) CDR3 of SEQ        ID NO: 20.

Even most preferably, the variable part of the light chain of thehumanized antibody according to the invention comprises, consistsessentially of or consists of the amino acid sequence of SEQ ID NO: 14;and the variable part of the heavy chain of the humanized antibodyaccording to the invention comprises, consists essentially of orconsists of the amino acid sequence of SEQ ID NO: 70; and the human IgGFc region comprises, consists essentially of or consists of the aminoacid sequence of SEQ ID NO: 74.

Even most preferably,

-   -   the variable part of the light chain of the humanized antibody        according to the invention comprises, consists essentially of or        consists of the amino acid sequence of SEQ ID NO: 14; and    -   the variable part of the heavy chain of the humanized antibody        according to the invention comprises, consists essentially of or        consists of the amino acid sequence of SEQ ID NO: 70; and    -   the human IgG Fc region comprises, consists essentially of or        consists of the amino acid sequence of SEQ ID NO: 74;    -   the variable part of the light chain of said antibody, which        comprises, consists essentially of or consists of the amino acid        sequence of SEQ ID NO: 14, comprises the CDR regions V_(L) CDR1        of SEQ ID NO: 12, V_(L) CDR2 SEQ ID NO: 9 and V_(L) CDR3 of SEQ        ID NO: 10; and    -   the variable part of the heavy chain of said antibody, which        comprises, consists essentially of or consists of the amino acid        sequence of SEQ ID NO: 70, comprises the CDR regions V_(H) CDR1        of SEQ ID NO: 25, V_(H) CDR2 SEQ ID NO: 71 and V_(H) CDR3 of SEQ        ID NO: 20.

Preferred polynucleotides of the present invention encode antibodies,comprising CDRs selected from those consisting of the amino acidsequences of SEQ ID NOs: 9, 10, 16, 29-31 and 37-39 in the light chainand selected from those consisting of the amino acid sequences of SEQ IDNOs: 20, 22, 23, 25, 26, 33-35, 41-43, 67, 69 and 71 in the heavy chain;and comprising a human IgG Fc region selected from SEQ ID NOs: 73 and74.

Further preferred are polynucleotides, which encode antibodies, whereinthe variable part of the light chain comprises, essentially consists orconsists of an amino acid sequence selected from SEQ ID NOs: 7, 11, 13,14, and 28; and wherein the human IgG Fc region comprises, consistsessentially of or consists of an amino acid sequence selected from SEQID NO: 73 or 74.

Even preferred are polynucleotides, which encode antibodies, wherein thevariable part of the heavy chain comprises, essentially consists orconsists of an amino acid sequence selected from SEQ ID NOs: 17, 21, 24,27, 32, 36, 40, 66, 68 and 70; and wherein the human IgG Fc regioncomprises, consists essentially of or consists of an amino acid sequenceselected from SEQ ID NO: 73 or 74.

The aforementioned polynucleotides can be integrated into expressionvectors well known in the art. Transfection of these expression vectorsin an appropriate host, the selection of the host as well as theexpression collection and purification of the light chains, heavychains, light/heavy chain dimers or intact antibodies, binding fragmentsor other immunoglobulin forms are well-known procedures in the art.

One skilled in the art can select a vector based on desired properties,for example, for production of a vector in a particular cell such as amammalian cell or a bacterial cell.

Any of a variety of inducible promoters or enhancers can be included inthe vector for expression of an antibody of the invention or nucleicacid that can be regulated. Such inducible systems, include, forexample, tetracycline inducible System (Gossen & Bizard, Proc. Natl.Acad. Sci. USA, 89:5547-5551 (1992); Gossen et al., Science,268:17664769 (1995); Clontech, Palo Alto, Calif.); metallothioneinpromoter induced by heavy metals; insect steroid hormone responsive toecdysone or related steroids such as muristerone (No et al., Proc. Natl.Acad. Sci. USA, 93:3346-3351 (1996); Yao et al., Nature, 366:476-479(1993); Invitrogen, Carlsbad, Calif.); mouse mammary tumor virus (MMTV)induced by steroids such as glucocorticoid and estrogen (Lee et al.,Nature, 294:228-232 (1981); and heat shock promoters inducible bytemperature changes; the rat neuron specific enolase gene promoter(Forss-Petter, et al., Neuron 5; 197-197 (1990)); the human β-actin genepromoter (Ray, et al., Genes and Development (1991) 5:2265-2273); thehuman platelet derived growth factor B (PDGF-B) chain gene promoter(Sasahara, et al., Cell (1991) 64:217-227); the rat sodium channel genepromoter (Maue, et al., Neuron (1990) 4:223-231); the human copper-zincsuperoxide dismutase gene promoter (Ceballos-Picot, et al., Brain Res.(1991) 552:198-214); and promoters for members of the mammalianPOU-domain regulatory gene family (Xi et al., (1989) Nature 340:35-42).

Regulatory elements, including promoters or enhancers, can beconstitutive or regulated, depending upon the nature of the regulation.The regulatory sequences or regulatory elements are operatively linkedto one of the polynucleotide sequences of the invention such that thephysical and functional relationship between the polynucleotide sequenceand the regulatory sequence allows transcription of the polynucleotidesequence. Vectors useful for expression in eukaryotic cells can include,for example, regulatory elements including the CAG promoter, the SV40early promoter, the cytomegalovirus (CMV) promoter, the mouse mammarytumor virus (MMTV) steroid-inducible promoter, Pgtf, Moloney marineleukemia virus (MMLV) promoter, thy-1 promoter and the like.

If desired, the vector can contain a selectable marker. As used herein,a “selectable marker” refers to a genetic element that provides aselectable phenotype to a cell in which the selectable marker has beenintroduced. A selectable marker is generally a gene whose gene productprovides resistance to an agent that inhibits cell growth or kills acell. A variety of selectable markers can be used in the DNA constructsof the invention, including, for example, Neo, Hyg, hisD, Gpt and Blegenes, as described, for example in Ausubel et al. (Current Protocols inMolecular Biology (Supplement 47), John Wiley & Sons, New York (1999))and U.S. Pat. No. 5,981,830. Drugs useful for selecting for the presenceof a selectable marker include, for example, G418 for Neo, hygromycinfor Hyg, histidinol for hisD, xanthine for Gpt, and bleomycin for Ble(see Ausubel et al, supra, (1999); U.S. Pat. No. 5,981,830). DNAconstructs of the invention can incorporate a positive selectablemarker, a negative selectable marker, or both (see, for example, U.S.Pat. No. 5,981,830). Various mammalian cell culture systems can also beemployed to express a recombinant protein. Examples of mammalianexpression systems include the COS-7 lines of monkey kidney fibroblasts,described by Gluzman, Cell, 23: 175 (1981). Other cell lines capable ofexpressing a compatible vector include, for example, the C127, 3T3, CHO,HeLa and BHK cell lines. Mammalian expression vectors will generallycomprise an origin of replication, a suitable promoter and enhancer, andalso any necessary ribosome binding sites, polyadenylation site, splicedonor and acceptor sites, transcriptional termination sequences, and 5′flanking nontranscribed sequences. DNA sequences derived from the SV40splice, and polyadenylation sites may be used to provide requirednontranscribed genetic elements.

The polypeptides can be recovered and purified from recombinant cellcultures by methods including ammonium sulfate or ethanol precipitation,acid extraction, anion or cation exchange chromatography,phosphocellulose chromatography, hydrophobic interaction chromatography,affinity chromatography, hydroxylapatite chromatography and lectinchromatography. Recovery can be facilitated if the polypeptide isexpressed at the surface of the cells, but such is not a prerequisite.Recovery may also be desirable of cleavage products that are cleavedfollowing expression of a longer form of the polypeptide. Proteinrefolding steps as known in this art can be used, as necessary, tocomplete configuration of the mature protein. High performance liquidchromatography (HPLC) can be employed for final purification steps.

Human constant region DNA sequences can be isolated in accordance withwell-known procedures from a variety of human cells.

The present invention pertains in particular to humanized antibodieswhich are characterized in that they bind to Aβ N3pE peptides with ahigh affinity. The present invention also pertains to antibodies whichare characterized in that they bind to Aβ N3pE peptides orimmunologically active fragments thereof with a high affinity. Said highaffinity means in the context of the present invention an affinity of aK_(D) value of 10⁻⁵ M, 10⁻⁶ M or 10⁻⁷ M or better, preferably a K_(D)value of 10⁻⁸ M or better, and even more preferably a K_(D) value of10⁻⁹ M−10⁻¹² M. Thereby, the inventive antibodies bind to monomeric AβN3pE with a higher affinity than previously known antibodies.

Preferably, the binding epitope of the humanized antibodies of thepresent invention in Aβ N3pE binds is an epitope, which carries apyroglutamate at the N-terminus. More preferably, the binding epitope ofthe humanized antibody of the invention is selected from the groupconsisting of

(SEQ ID NO: 50) pEFRHDSGYEVHHQKLV, (SEQ ID NO: 54) pEFRHDSGYEVHHQKL,(SEQ ID NO: 55) pEFRHDSGYEVHHQK, (SEQ ID NO: 56) pEFRHDSGYEVHHQ, (SEQ IDNO: 57) pEFRHDSGYEVHH, (SEQ ID NO: 58) pEFRHDSGYEVH, (SEQ ID NO: 59)pEFRHDSGYEV, (SEQ ID NO: 60) pEFRHDSGYE, (SEQ ID NO: 61) pEFRHDSGY, (SEQID NO: 62) pEFRHDSG, (SEQ ID NO: 63) pEFRHDS, (SEQ ID NO: 72) pEFRHD(SEQ ID NO: 64) pEFRH, and (SEQ ID NO: 65) pEFR.

Most preferably, the humanized antibodies of the invention do not bindto binding epitopes that do not carry a pyroglutamate at the N-terminus.

Even most preferably, when binding to the aforementioned andsubsequently mentioned binding epitopes, the humanized antibodies of theinvention always bind to sequences or parts of sequences, which containthe pyroglutamate at the N-terminus. The humanized antibodies of theinvention do not bind to sequences or parts of sequences, which do notcontain the pyroglutamate at the N-terminus.

Further, the humanized antibody of the invention can also bind to an AβN3pE variant.

In the context of the present invention, an Aβ N3pE variant is inparticular

-   -   pE-Aβ₃₋₃₈,    -   pE-Aβ₃₋₄₀,    -   pE-Aβ₃₋₄₂

Further variants of Aβ N3pE peptides are all Aβ N3pE variants, whichhave been shown to accumulate in the brain as a consequence ofAlzheimer's disease or preceding Alzheimer's disease. These are thepE-Aβ_(3-x) peptides, wherein x is defined as an integer having a valuein the range of 19 to 42, e.g. in the above pE-Aβ₃₋₄₂, “42” would be theinteger for “x”.

In the context of the present invention a “functional variant” of theinventive humanized antibody is an antibody which retains the bindingcapacities, in particular binding capacities with high affinity to apE-Aβ_(3-x) peptide. The provision of such functional variants is knownin the art and encompasses the above-mentioned possibilities, which wereindicated under the definition of antibodies and fragments thereof.

In a further embodiment, the humanized antibody is an antibody fragment,as defined above.

In a further preferred embodiment, the humanized antibody of theinvention is a humanized antibody which has thecomplementarity-determining regions (CDRs) of the above-definedantibodies. Preferably, the antibody can be labeled; possible labels arethose as mentioned above and all those known to a person skilled in theart of diagnostic uses of antibodies in particular.

In another embodiment, the humanized antibodies may be immobilized on asolid phase.

In another embodiment, the humanized antibodies according to theinvention and as described herein before or a fragments thereof, exhibita binding affinity to an Aβ N3pE oligomer, fiber, fibril or filamentwhich is at least 2 times, particularly at least 4 times, particularlyat least 10 times, particularly at least 15 times, more particularly atleast 20 times, but especially at least 25 times higher than the bindingaffinity to an Aβ N3pE monomer.

In still another embodiment, humanized antibodies or fragments thereofare provided as described herein before, which substantially bind toaggregated Aβ, including Aβ plaques, which contain Aβ N3pE, in themammalian, particularly the human brain but, preferably, do not show anysignificant cross-reactivity with amyloid precursor protein (APP).

In another aspect of the invention, humanized antibodies or fragmentsthereof are provided as described herein before, which antibodiessubstantially bind to oligomeric or polymeric amyloid, which contains AβN3pE, particularly amyloid β (Aβ) in the mammalian, particularly thehuman brain but, preferably, do not show any significantcross-reactivity with amyloid precursor protein (APP).

The present invention relates also to compositions comprising saidhumanized antibodies and the use of said compositions for the treatmentof amyloidosis, especially for the treatment of neurodegenerativedisease in a mammal, in particular in a human. Said neurodegenerativedisease is in particular selected from the group consisting of mildcognitive impairment (MCI), Alzheimer's disease (AD), like for instancesporadic Alzheimer's disease (SAD) or Familial Alzheimer's dementias(FAD) like Familial British Dementia (FBD) and Familial Danish Dementia(FDD), neurodegeneration in Down Syndrome. Preferably, saidneurodegenerative disease is Alzheimer's disease.

Thus, in a preferred embodiment, the present invention is directed to amethod of treating and/or preventing conditions characterized by theformation of plaques comprising Aβ N3pE in mammals, preferably inhumans, which method comprises administering, preferably peripherally,to a human in need of such treatment a therapeutically orprophylactically effective amount of a humanized monoclonal antibody ofthe invention or a immunologically reactive fragment thereof, whichantibody specifically binds to an epitope of the Aβ N3pE peptide thatcarries pyroglutamate at the N-terminus.

In another embodiment, the invention is directed to a method to inhibitthe formation of amyloid plaques and to clear or remove amyloid plaquesin mammals, preferably in humans, which method comprises administeringto a human subject in need of such inhibition an effective amount of ahumanized antibody that binds to Aβ N3pE in the circulation, body fluidsor tissues, especially in the brain and further preferably, leads to theclearance of Aβ N3pE in plasma and the brain.

Accordingly, the invention also provides methods of reversing cognitivedecline, improving cognition, treating cognitive decline, and preventingcognitive decline in a subject diagnosed with mild cognitive impairment(MCI), Alzheimer's disease (AD), like for instance sporadic Alzheimer'sdisease (SAD) or Familial Alzheimer's dementias (FAD) like FamilialBritish Dementia (FBD) and Familial Danish Dementia (FDD),neurodegeneration in Down Syndrome and clinical or pre-clinical cerebralamyloid angiopathy, preferably Alzheimer's disease comprisingadministering to the subject an effective amount of a humanized antibodyof the invention.

The invention also provides the use of a humanized antibody of theinvention for the manufacture of a medicament, for treating, preventing,or reversing mild cognitive impairment (MCI), Alzheimer's disease (AD),like for instance sporadic Alzheimer's disease (SAD) or FamilialAlzheimer's dementias (FAD) like Familial British Dementia (FBD) andFamilial Danish Dementia (FDD), neurodegeneration in Down Syndrome andclinical or pre-clinical cerebral amyloid angiopathy, preferablyAlzheimer's disease; or to reverse cognitive decline, improve cognition,treat cognitive decline, and prevent cognitive decline in a subjectdiagnosed with mild cognitive impairment (MCI), Alzheimer's disease(AD), like for instance sporadic Alzheimer's disease (SAD) or FamilialAlzheimer's dementias (FAD) like Familial British Dementia (FBD) andFamilial Danish Dementia (FDD), neurodegeneration in Down Syndrome andclinical or pre-clinical cerebral amyloid angiopathy, preferablyAlzheimer's disease.

The invention further provides the humanized antibodies disclosed hereinfor use in the prevention, treatment, or the reversion of mild cognitiveimpairment (MCI), Alzheimer's disease (AD), like for instance sporadicAlzheimer's disease (SAD) or Familial Alzheimer's dementias (FAD) likeFamilial British Dementia (FBD) and Familial Danish Dementia (FDD),neurodegeneration in Down Syndrome and clinical or pre-clinical cerebralamyloid angiopathy, preferably Alzheimer's disease; for treating,preventing, or the reversion of cognitive decline, improvement ofcognition, treatment of cognitive decline, and prevention of cognitivedecline in a subject diagnosed with mild cognitive impairment (MCI),Alzheimer's disease (AD), like for instance sporadic Alzheimer's disease(SAD) or Familial Alzheimer's dementias (FAD) like Familial BritishDementia (FBD) and Familial Danish Dementia (FDD), neurodegeneration inDown Syndrome and clinical or pre-clinical cerebral amyloid angiopathy,preferably Alzheimer's disease; or the inhibition of the formation ofan1yloid plaques or the effects of Aβ N3pE in mammals, preferably inhumans.

In a specific embodiment the invention provides a method for retainingor increasing cognitive memory capacity but, particularly, for restoringthe cognitive memory capacity of a mammal, particularly a human,suffering from memory impairment by administering to an animal,particularly a mammal or a human, a humanized antibody, or apharmaceutical composition comprising a humanized antibody according tothe invention and as described herein before.

The invention further provides methods to assess the response of a humansubject to treatment with a humanized antibody that binds Aβ N3pE or avariant thereof, comprising:

a) administering a humanized antibody of the invention or a fragmentthereof to the subject; andb) measuring the concentration of Aβ N3pE in a biological sample takenfrom the subject.

The invention also provides a method of treating a human subject with anantibody that binds Aβ N3pE or a variant thereof, comprising:

a) administering a first amount of the antibody or fragment thereof tothe subject;b) within 3 hours to two weeks after administering the first dose,measuring the concentration of Aβ N3pE in a biological sample taken fromthe subject;c) if necessary, calculating a second amount of antibody or fragmentthereof based on the result of step b), which second amount is the sameas or different than the first amount; andd) administering the second amount of the antibody or fragment.

The invention also includes a method of assessing in a mammalian,preferably a human subject the efficacy of an antibody that binds to AβN3pE, or a fragment thereof, for inhibiting or preventing Aβ N3pErelated amyloid plaque formation, for reducing the load of Aβ N3pEcontaining plaques, for reducing the effects of toxic Aβ N3pE andvariants thereof, or for treating a condition or a disease associatedwith plaques containing Aβ N3pE, comprising:

a) obtaining a first biological sample form the subject;b) measuring a baseline concentration of Aβ N3pE in the first sample;c) administering a humanized antibody of the invention or fragmentthereof to the subject;d) within 3 hours to two weeks after administering the antibody orfragment thereof, obtaining a second biological sample from the subject;ande) measuring the concentration of Aβ N3pE in the second biologicalsample; wherein, efficacy is related to the quantity of Aβ N3pE bound tothe antibody in the blood and the concentration of Aβ N3pE, inparticular the reduction of the concentration thereof, in the secondbiological sample compared to the first biological sample.

The biological sample may be any sample, for example from a human. Inone specific example, the sample is a tissue sample, a body fluid sampleor a cell sample. In one embodiment, the biological sample is selectedfrom the group consisting of blood, serum, urine, cerebrospinal fluid(CSF), plasma, lymph, saliva, sweat, pleural fluid, synovial fluid, tearfluid, bile and pancreas secretion. In a further embodiment, thebiological sample is plasma. In a preferred embodiment, the biologicalsample is CSF.

The biological sample can be obtained from a subject in a mannerwell-known to a person skilled in the art. In particular, a blood samplecan be obtained from a subject and the blood sample can be separatedinto serum and plasma by conventional methods. The subject, from whichthe biological sample is obtained is preferably a subject suspected ofbeing afflicted with a disease or condition of amyloidosis, preferablyAlzheimer's disease, at risk of developing Alzheimer's disease and/orbeing at risk of or having any other kind of dementia.

In particular, the sample is obtained from a subject suspected of havingMild Cognitive Impairment (MCI) and/or being in the early stages ofAlzheimer's disease.

The efficacy of the humanized antibodies of the invention in thediagnosis, prevention and/or treatment of amyloidosis, such as mildcognitive impairment, Alzheimer's Disease, Familial British Dementia orFamilial Danish Dementia and, e.g. neurodegeneration in Down Syndromecan be tested in existing animal models of Alzheimer's disease.

Suitable animal models of Alzheimer's Disease are reviewed in McGowan etal. TRENDS in Genetics, Vol. 22, No. May 2006, pp 281-289, and areselected from PDAPP, Tg2576, APP23, TgCRND8, PSEN_(1M146)V orPSEN_(1M146L), PSAPP, APP_(Dutch), BRI-Aβ40 and BRI-Aβ42, JNPL3,Tau_(P301S), Tau_(V337M), Tau_(R406W), rTg4510, H_(tau), TAPP, 3×TgAD,as described below.

PDAPP: First mutant APP transgenic model with robust plaque pathology.Mice express a human APP cDNA with the Indiana mutation (APP_(V717F)).Plaque pathology begins between 6-9 months in hemizygous PDAPP mice.There is synapse loss but no overt cell loss and not NFT pathology isobserved. This model has been used widely in vaccination therapystrategies.

Tg2576: Mice express mutant APP_(SWE) under control of the hamster prionpromoter. Plaque pathology is observed from 9 months of age. These micehave cognitive deficits but no cell loss or NFT pathology. This model isone of the most widely used transgenic models in the field ofAlzheimer's disease.

APP23: Mice express mutant APP_(SWE) under control of the Thy1 promoter.Prominent cerebrovascular amyloid, amyloid deposits are observed from 6months of age and some hippocampal neuronal loss is associated withamyloid plaque formation.

TgCRND8: Mice express multiple APP mutations (Swedish plus Indiana).Cognitive deficits coincide with rapid extracellular plaque developmentat 3 months of age. The cognitive deficits can be reversed by Aβvaccination therapy.

PSEN_(1M146V) or PSEN_(1M146L) (lines 6.2 and 8.9, respectively): Thesemodels where the first demonstration in vivo that mutant PSEN1selectively elevates Aβ42. No overt plaque pathology is observed.

PSAPP (Tg2576×PSEN_(1M146L), PSEN1-A246E+APP_(SWE)): Bigenic transgenicmice, with the addition of the mutant PSEN1 transgene which markedlyaccelerated amyloid pathology compared with singly transgenic mutant APPmice, demonstrating that the PSEN1-driven elevation of Aβ 42 enhancesplaque pathology.

APP_(Dutch): Mice express APP with the Dutch mutation that causeshereditary cerebral hemorrhage with amyloidosis-Dutch type in humans.APP_(Dutch) mice develop severe congophilic amyloid angiopathy. Theaddition of a mutant PSEN1 transgene redistributes the amyloid pathologyto the parenchyma indicating differing roles for Aβ 40 and Aβ 42 invascular and parenchymal amyloid pathology.

BRI-Aβ40 and BRI-Aβ42: Mice express individual Aβ isoforms without APPover-expression. Only mice expressing Aβ 42 develop senile plaques andCAA, whereas BRI-Aβ 40 mice do not develop plaques, suggesting that Aβ42 is essential for plaque formation.

JNPL3: Mice express 4R0N MAPT with the P301L mutation. This is the firsttransgenic model, with marked tangle pathology and cell loss,demonstrating that MAPT alone can cause cellular damage and loss. JNPL3mice develop motor impairments with age owing to server pathology andmotor neutron loss in the spinal cord.

Tau_(P301S): Tansgenic mice expressing the shortest isoform of 4R MAPTwith the P301S mutation. Homozygous mice develop severe paraparesis at5-6 months of age with widespread neurofibrillary pathology in the brainand spinal cord and neuronal loss in the spinal cord.

Tau_(V337M): Low level synthesis of 4R MAPT with the V337M mutation(1/10 endogenous MAPT) driven by the promoter of platelet-derived growthfactor (PDGF). The development of neurofibrillary pathology in thesemice suggests the nature of the MAPT rather than absolute MAPTintracellular concentration drives pathology.

Tau_(R406W): Mice expressing 4R human MAPT with the R406W mutation undercontrol of the CAMKII promoter. Mice develop MAPT inclusions in theforebrain from 18 months of age and have impaired associative memory.

rTg4510: Inducible MAPT transgenic mice using the TET-off system.Abnormal MAPT pathology occurs from one month of age. Mice haveprogressive NFT pathology and severe cell loss. Cognitive deficits areevident from 2.5 months of age. Turning off the transgene improvescognitive performance but NT pathology worsens.

H_(tau): Transgenic mice expressing human genomic MAPT only (mouse MAPTknocked-out). Htau mice accumulate hyperphosphorylated MAPT from 6months and develop Thio-S-positive NFT by the time they are 15 monthsold.

TAPP (Tg2576×JNPL3): Increased MAPT forebrain pathology in TAPP micecompared with JNPL3 suggesting mutant APP and/or Aβ can affectdownstream MAPT pathology.

3×TgAD: Triple transgenic model expressing mutant APP_(SWE),MAPT_(P301L) on a PSEN1_(M146V) ‘knock-in’ background (PSNE1-KI). Micedevelop plaques from 6 months and MAPT pathology from the time they are12 months old, strengthening the hypothesis that APP or Aβ can directlyinfluence neurofibrillary pathology.

Moreover, WO 2009/034158 discloses non-human transgenic animal models,wherein the transgene encodes at least one amyloid beta (Aβ) peptideselected from the group consisting of AβN3E-42, AβN3Q-42, AβN3E-40 andAβN3Q-40. These Aβ peptides are substrates of QC and QPCTL, resulting inthe cyclization of the N-terminal glutamine (Q) or glutamate (N) topyroglutamate (pGlu). Thus, these transgenic animal models provide amodel system for the investigation of the effect of pGlu-Aβ peptides onthe course of the development of neurodegenration.

Anti-Aβ pN3pE antibodies may also be useful in diagnostic assays for AβpN3pE, e.g. detecting its occurrence in specific cells, tissues, orserum. Thus, The humanized antibodies according to the present inventionare especially useful in a diagnostic method to detect amyloidosis, inparticular a neurodegenerative disease selected from the groupconsisting of mild cognitive impairment (MCI), Alzheimer's disease (AD),like for instance sporadic Alzheimer's disease (SAD) or FamilialAlzheimer's dementias (FAD) like Familial British Dementia (FBD) andFamilial Danish Dementia (FDD), neurodegeneration in Down Syndrome;preferably Alzheimer's disease.

For diagnostic applications, the antibody typically will be labelledwith a detectable moiety. Numerous labels are available which can begenerally grouped into the following categories:

(a) Radioisotopes, such as ³⁵S, ¹⁴C, ¹²⁵I, ³H, and ¹³¹I. The antibodycan be labeled with the radioisotope using the techniques described inCurrent Protocols in Immunology, Volumes 1 and 2, Gütigen et al., Ed.,Wiley-Interscience. New York, N.Y. Pubs., (1991) for example andradioactivity can be measured using scintillation counting.(b) Fluorescent labels such as rare earth chelates (europium chelates)or fluorescein and its derivatives, rhodamine and its derivatives,dansyl, Lissamine, p hycoerythrin and Texas Red are available. Thefluorescent labels can be conjugated to the antibody using thetechniques disclosed in Current Protocols in Immunology, supra forexample. Fluorescence can be quantified using a fluorimeter.(c) Various enzyme-substrate labels are available. The enzyme generallycatalyses a chemical alteration of the chromogenic substrate which canbe measured using various techniques. For example, the enzyme maycatalyze a color change in a substrate, which can be measuredspectrophotometrically. Alternatively, the enzyme may alter thefluorescence or chemiluminescence of the substrate. Techniques forquantifying a change in fluorescence are described above. Thechemiluminescent substrate becomes electronically excited by a chemicalreaction and may then emit light which can be measured (using achemiluminometer, for example) or donates energy to a fluorescentacceptor. Examples of enzymatic labels include luciferases (e.g, fireflyluciferase and bacterial luciferase; U.S. Pat. No. 4,737,456),luciferin, 2,3-dihydrophthalazinediones, malate dehydrogenase, urease,peroxidase such as horseradish peroxidase (HRPO), alkaline phosphatase.0-galactosidase, glucoamylase, lysozyme, saccharide oxidases (e.g.,glucose oxidase, galactose oxidase, and glucose-6-phosphatedehydrogenase), heterocyclic oxidases (such as uricase and xanthineoxidase), lactoperoxidase, microperoxidase, and the like. Techniques forconjugating enzymes to antibodies are described in O'Sullivan et al.,Methods for the Preparation of Enzyme-Antibody Conjugates for use inEnzyme Immunoassay, in Methods in Enzym (ed Langone & H. Van Vunakis),Academic Press, New York, 73: 147-166 (1981).

Examples of enzyme-substrate combinations include, for example:

(i) Horseradish peroxidase (HRPO) with hydrogen peroxidase as asubstrate, wherein the hydrogen peroxidase oxidizes a dye precursor(e.g. orthophenylene diamine (OPD) or 3,3′,5,5′-tetramethyl benzidinehydrochloride (TMB));(ii) alkaline phosphatase (Aβ) with para-Nitrophenyl phosphate aschromogenic substrate; and(iii) β-D-galactosidase (β-D-Gal) with a chromogenic substrate (e.g.p-nitrophenyl-β-D-galactosidase) or the fluorogenic substrate4-methylumbelliferyl-β-D-galactosidase.

Numerous other enzyme-substrate combinations are available to thoseskilled in the art.

Sometimes, the label is indirectly conjugated with the antibody. Theskilled artisan will be aware of various techniques for achieving this.For example, the antibody can be conjugated with biotin and any of thethree broad categories of labels mentioned above can be conjugated withavidin, or vice versa. Biotin binds selectively to avidin and thus, thelabel can be conjugated with the antibody in this indirect manner.Alternatively, to achieve indirect conjugation of the label with theantibody, the antibody is conjugated with a small hapten (e.g. digoxin)and one of the different types of labels mentioned above is conjugatedwith an anti-hapten antibody (e.g. anti-digoxin antibody). Thus,indirect conjugation of the label with the antibody can be achieved.

The humanized antibodies of the present invention may be employed in anyknown assay method, such as competitive binding assays, direct andindirect sandwich assays, and immunoprecipitation assays. Zola,Monoclonal Antibodies A Manual of Techniques, pp. 147-158 (CRC Press.Inc., 1987) Competitive binding assays rely on the ability of a labeledstandard to compete with the test sample analyte for binding with alimited amount of antibody. The amount of Aβ N3pE in the test sample isinversely proportional to the amount of standard that becomes bound tothe antibodies. To facilitate determining the amount of standard thatbecomes bound, the antibodies generally are insolubilized before orafter the competition, so that the standard and analyte that are boundto the antibodies may conveniently be separated from the standard andanalyte which remain unbound.

Sandwich assays involve the use of two antibodies, each capable ofbinding to a different immunogenic portion, or epitope, of the proteinto be detected. In a sandwich assay, the test sample analyte is bound bya first antibody which is immobilized on a solid support, and thereaftera second antibody binds to the analyte, thus forming an insolublethree-part complex. The second antibody may itself be labeled with adetectable moiety (direct sandwich assays) or may be measured using ananti-immunoglobulin antibody that is labeled with a detectable moiety(indirect sandwich assay). For example, one preferable type of sandwichassay is an ELISA assay, in which case the detectable moiety is anenzyme.

For immunohistochemistry, the tissue sample may be fresh or frozen ormay be embedded in paraffin and fixed with a preservative such asformalin, for example.

The present invention also relates to a composition which comprises thehumanized antibodies as defined above, wherein said composition is acomposition for a diagnostic use, especially for the diagnosis of aneurodegenerative disease selected from the group consisting of mildcognitive impairment (MCI), Alzheimer's disease (AD), like for instancesporadic Alzheimer's disease (SAD) or Familial Alzheimer's dementias(FAD) like Familial British Dementia (FBD) and Familial Danish Dementia(FDD), neurodegeneration in Down Syndrome; preferably Alzheimer'sdisease; in particular by detection of Aβ N3pE or variants thereof in abiological sample.

Diagnostic Kits

As a matter of convenience, the antibody of the present invention can beprovided in a kit, i.e., a packaged combination of reagents inpredetermined amounts with instructions for performing the diagnosticassay. Where the antibody is labelled with an enzyme, the kit willinclude substrates and cofactors required by the enzyme (e.g. asubstrate precursor which provides the detectable chromophore orfluorophore). In addition, other additives may be included such asstabilizers, buffers (e.g. a block buffer or lysis buffer) and the like.The relative amounts of the various reagents may be varied widely toprovide for concentrations in solution of the reagents whichsubstantially optimize the sensitivity of the assay. Particularly, thereagents may be provided as dry powders, usually lyophilized, includingexcipients which on dissolution will provide a reagent solution havingthe appropriate concentration.

The diagnostic kit according to the invention may contain a furtherbiologically active substance as described below. Especially preferredfor the use in the diagnostic kit as said further biologically activesubstance is an inhibitor of glutaminyl cyclase.

The diagnostic kit of the invention is especially useful for thedetection and diagnosis of amyloid-associated diseases and conditions,in particular neurodegenerative diseases selected from the groupconsisting of mild cognitive impairment (MCI), Alzheimer's disease (AD),like for instance sporadic Alzheimer's disease (SAD) or FamilialAlzheimer's dementias (FAD) like Familial British Dementia (FBD) andFamilial Danish Dementia (FDD), neurodegeneration in Down Syndrome;preferably Alzheimer's disease.

The present invention also pertains to the humanized antibody of theinvention or the composition comprising the humanized antibody, both asdefined above, for use in an in vitro diagnostic method. In particular,this diagnostic method is directed to diagnosis of a neurodegenerativedisease selected from the group consisting of mild cognitive impairment(MCI), Alzheimer's disease (AD), like for instance sporadic Alzheimer'sdisease (SAD) or Familial Alzheimer's dementias (FAD) like FamilialBritish Dementia (FBD) and Familial Danish Dementia (FDD),neurodegeneration in Down Syndrome; preferably Alzheimer's disease;especially by detecting an Aβ N3pE or variants thereof in a biologicalsample.

In a particularly preferred embodiment, the present invention pertainsto the following method:

In vitro or in situ diagnostic method for the diagnosis of anamyloid-associated disease or condition, preferably Alzheimer's disease,comprising the following steps:

contacting a humanized antibody according to the invention with asample, preferably selected from a serum, liquor or CSF sample, mostpreferably a serum sample; or a specific body part or body area of asubject suspected to be afflicted with said condition or disease, anddetecting binding of the antibody to Aβ N3pE, from the sample.

More particularly, the invention relates to a method of diagnosis of anamyloid-associated disease or condition, preferably Alzheimer's disease,comprising detecting the immunospecific binding of a humanized antibodyof the invention or an immunologically active fragment thereof to AβN3pE, in a sample or in situ which includes the steps of

(a) bringing the sample or a specific body part or body area suspectedto contain the amyloid protein into contact with a humanized antibody ofthe invention, or a fragment thereof;(b) allowing the antibody and/or a functional part thereof, to bind toAβ N3pE to form an immunological complex;(c) detecting the formation of the immunological complex; and(d) correlating the presence or absence of the immunological complexwith the presence or absence of Aβ N3pE in the sample or specific bodypart or area.

Also comprised is a method of determining the extent of amyloidogenicplaque burden in a tissue and/or body fluids comprising

(a) obtaining a sample representative of the tissue and/or body fluidsunder investigation;(b) testing said sample for the presence of amyloid protein with ahumanized antibody according to the invention, or a chimeric antibody ora fragment thereof;(c) determining the amount of humanized antibody bound to the protein;and(d) calculating the plaque burden in the tissue and/or body fluids.

In particular, the invention relates to a method of determining theextent of amyloidogenic plaque burden in a tissue and/or body fluids,wherein the formation of the immunological complex in step c) isdetermined such that presence or absence of the immunological complexcorrelates with presence or absence of amyloid protein, in particular AβN3pE.

In still another embodiment, the invention relates to a compositioncomprising the humanized antibody according to the invention, or achimeric antibody or a fragment thereof, and as described herein beforeincluding any functionally equivalent antibody or any derivative orfunctional parts thereof, in particular a composition which is apharmaceutical composition optionally further comprising apharmaceutically acceptable carrier.

In another embodiment of the invention, said composition comprises thehumanized antibody in a therapeutically effective amount.

Further comprised by the invention is a mixture comprising a humanizedantibody of the invention, or a chimeric antibody or a fragment thereof,and as described herein before including any functionally equivalentantibody or any derivative or functional parts thereof, in atherapeutically effective amount and, optionally, a further biologicallyactive substance and/or a pharmaceutically acceptable carrier and/or adiluent and/or an excipient.

In particular, the invention relates to a mixture, wherein the furtherbiologically active substance is a compound used in the medication ofamyloidosis, a group of diseases and disorders associated with amyloidor amyloid-like protein such as Aβ N3pE involved in neurodegenerativediseases selected from the group consisting of mild cognitive impairment(MCI), Alzheimer's disease (AD), like for instance sporadic Alzheimer'sdisease (SAD) or Familial Alzheimer's dementias (FAD) like FamilialBritish Dementia (FBD) and Familial Danish Dementia (FDD),neurodegeneration in Down Syndrome; preferably Alzheimer's disease.

In another embodiment of the invention, the other biologically activesubstance or compound may also be a therapeutic agent that may be usedin the treatment of amyloidosis caused by Aβ N3pE or may be used in themedication of other neurological disorders.

The other biologically active substance or compound may exert itsbiological effect by the same or a similar mechanism as the antibodyaccording to the invention or by an unrelated mechanism of action or bya multiplicity of related and/or unrelated mechanisms of action.

Generally, the other biologically active compound may includeneutron-transmission enhancers, psychotherapeutic drugs, acetylcholineesterase inhibitors, calcium-channel blockers, biogenic amines,benzodiazepine tranquilizers, acetylcholine synthesis, storage orrelease enhancers, acetylcholine postsynaptic receptor agonists,monoamine oxidase-A or -B inhibitors, N-methyl-D-aspartate glutamatereceptor antagonists, non-steroidal anti-inflammatory drugs,antioxidants, and serotonergic receptor antagonists.

More particularly, the invention relates to a mixture comprising atleast one compound selected from the group consisting of compoundseffective against oxidative stress, anti-apoptotic compounds, metalchelators, inhibitors of DNA repair such as pirenzepin and metabolites,3-amino-1-propanesulfonic acid (3 APS), 1,3-propanedisulfonate (1,3PDS),α-secretase activators, β- and γ-secretase inhibitors, tau proteins,neurotransmitter, _(/)β-sheet breakers, attractants for amyloid betaclearing/depleting cellular components, inhibitors of N-terminaltruncated amyloid beta including pyroglutamated amyloid beta β-42, suchas inhibitors of glutaminyl cyclase, anti-inflammatory molecules, orcholinesterase inhibitors (ChEIs) such as tacrine, rivastigmine,donepezil, and/or galantamine, MI agonists and other drugs including anyamyloid or tau modifying drug and nutritive supplements, and nutritivesupplements, together with an antibody according to the presentinvention and, optionally, a pharmaceutically acceptable carrier and/ora diluent and/or an excipient.

The invention further relates to a mixture, wherein the compound is acholinesterase inhibitor (ChEIs), particularly a mixture, wherein thecompound is one selected from the group consisting of tacrine,rivastigmine, donepezil, galantamine, niacin and memantine.

In a further embodiment, the mixtures according to the invention maycomprise niacin or memantine together with an antibody according to thepresent invention and, optionally, a pharmaceutically acceptable carrierand/or a diluent and/or an excipient.

In a further embodiment, the mixtures according to the invention maycomprise a glutaminyl cyclase inhibitor together with an antibodyaccording to the present invention and, optionally, a pharmaceuticallyacceptable carrier and/or a diluent and/or an excipient.

Preferred inhibitors of glutaminyl cyclase are described in WO2005/075436, WO 2008/055945, WO 2008/055947, WO 2008/055950, WO2008/065141, WO 2008/110523, WO 2008/128981, WO 2008/128982, WO2008/128983, WO 2008/128984, WO 2008/128985, WO 2008/128986, WO2008/128987, WO 2010/026212, WO 2011/131748, WO 2011/029920, WO2011/107530, WO 2011/110613, WO 2012/123563 and WO 2014/140279, thedisclosure of which is incorporated herein by reference.

In still another embodiment of the invention mixtures are provided thatcomprise “atypical antipsychotics” such as, for example clozapine,ziprasidone, risperidone, aripiprazole or olanzapine for the treatmentof positive and negative psychotic symptoms including hallucinations,delusions, thought disorders (manifested by marked incoherence,derailment, tangentiality), and bizarre or disorganized behavior, aswell as anhedonia, flattened affect, apathy, and social withdrawal,together with an antibody, particularly a monoclonal antibody accordingto the invention, but particularly a chimeric antibody or a fragmentthereof, or a humanized antibody or a fragment thereof according to theinvention and as described herein and, optionally, a pharmaceuticallyacceptable carrier and/or a diluent and/or an excipient.

In a specific embodiment of the invention, the compositions and mixturesaccording to the invention and as described herein before comprise thehumanized antibody of the invention and the biologically activesubstance, respectively, in a therapeutically effective amount.

Other compounds that can be suitably used in mixtures in combinationwith the humanized antibody according to the present invention aredescribed in WO2008/065141 (see especially pages 37/38), includingPEP-inhibitors (pp. 43/44), LiCl, inhibitors of dipeptidylaminopeptidases, preferably inhibitors of DP IV or DP IV-like enzymes(see pp. 48/49); acetylcholinesterase (ACE) inhibitors (see p. 47), PIMTenhancers, inhibitors of beta secretases (see p. 41), inhibitors ofgamma secretases (see pp. 41/42), inhibitors of neutral endopeptidase,inhibitors of phosphodiesterase-4 (PDE-4) (see pp. 42/43), TNFalphainhibitors, muscarinic M1 receptor antagonists (see p. 46), NMDAreceptor antagonists (see pp. 47/48), sigma-1 receptor inhibitors,histamine H3 antagonists (see p. 43), immunomodulatory agents,immunosuppressive agents or an agent selected from the group consistingof antegren (natalizumab), Neurelan (fampridine-SR), campath(alemtuzumab), IR208, NBI 5788/MSP 771 (tiplimotide), paclitaxel,Anergix.MS (AG 284), SH636, Differin (CD 271, adapalene), BAY 361677(interleukin-4), matrix-metalloproteinase-inhibitors (e.g. BB 76163),interferon-tau (trophoblastin) and SAIK-MS; beta-amyloid antibodies (seep. 44), cysteine protease inhibitors (see p. 44); MCP-1 antagonists (seepp. 44/45), amyloid protein deposition inhibitors (see 42) and betaamyloid synthesis inhibitors (see p. 42), which document is incorporatedherein by reference.

In another embodiment, the invention relates to a mixture comprising thehumanized antibody according to the invention, or a chimeric antibody ora fragment thereof and as described herein before and/or thebiologically active substance in a therapeutically effective amount.

The pharmaceutical compositions may be formulated with pharmaceuticallyacceptable carriers or diluents as well as any other known adjuvants andexcipients in accordance with conventional techniques such as thosedisclosed in Remington: The Science and Practice of Pharmacy, 21thEdition, Gennaro, Ed., Mack Publishing Co., Easton, Pa., 2005.

The pharmaceutically acceptable carriers or diluents as well as anyother known adjuvants and excipients should be suitable for the chosenhumanized antibody of the present invention and the chosen mode ofadministration. Suitability for carriers and other components ofpharmaceutical compositions is determined based on the lack ofsignificant negative impact on the desired biological properties of thechosen humanized antibody or pharmaceutical composition of the presentinvention (e.g., less than a substantial impact (10% or less relativeinhibition, 5% or less relative inhibition, etc.)) on antigen binding.

A pharmaceutical composition of the present invention may also includediluents, fillers, salts, buffers, detergents (e.g., a nonionicdetergent, such as Tween-20 or Tween-80), stabilizers (e.g., sugars orprotein-free amino acids), preservatives, tissue fixatives,solubilizers, and/or other materials suitable for inclusion in apharmaceutical composition.

The actual dosage levels of the active ingredients in the pharmaceuticalcompositions of the present invention may be varied so as to obtain anamount of the active ingredient which is effective to achieve thedesired therapeutic response for a particular patient, composition, andmode of administration. The selected dosage level will depend upon avariety of pharmacokinetic factors including the activity of theparticular compositions of the present invention employed, or the amidethereof, the route of administration, the time of administration, therate of excretion of the particular humanized antibody being employed,the duration of the treatment, other drugs, compounds and/or materialsused in combination with the particular compositions employed, the age,sex, weight, condition, general health and prior medical history of thepatient being treated, and like factors well known in the medical arts.

The pharmaceutical composition may be administered by any suitable routeand mode. Suitable routes of administering a humanized antibody of thepresent invention in vivo and in vitro are well known in the art and maybe selected by those of ordinary skill in the art.

In one embodiment, a pharmaceutical composition of the present inventionis administered parenterally.

The phrases “parenteral administration” and “administered parenterally”as used herein means modes of administration other than enteral andtopical administration, usually by injection, and include epidermal,intravenous, intramuscular, intraarterial, intrathecal, intracapsular,intraorbital, intracardiac, intradermal, intraperitoneal,intratendinous, transtracheal, subcutaneous, subcuticular,intraarticular, subcapsular, subarachnoid, intraspinal, intracranial,intrathoracic, epidural and intrasternal injection and infusion.

In one embodiment that pharmaceutical composition is administered byintravenous or subcutaneous injection or infusion.

Pharmaceutically acceptable carriers include any and all suitablesolvents, dispersion media, coatings, antibacterial and antifungalagents, isotonicity agents, antioxidants and absorption delaying agents,and the like that are physiologically compatible with a humanizedantibody of the present invention.

Examples of suitable aqueous and nonaqueous carriers which may beemployed in the pharmaceutical compositions of the present inventioninclude water, saline, phosphate buffered saline, ethanol, dextrose,polyols (such as glycerol, propylene glycol, polyethylene glycol, andthe like), and suitable mixtures thereof, vegetable oils, such as oliveoil, corn oil, peanut oil, cottonseed oil, and sesame oil, carboxymethylcellulose colloidal solutions, tragacanth gum and injectable organicesters, such as ethyl oleate, and/or various buffers. Other carriers arewell known in the pharmaceutical arts.

Pharmaceutically acceptable carriers include sterile aqueous solutionsor dispersions and sterile powders for the extemporaneous preparation ofsterile injectable solutions or dispersion. The use of such media andagents for pharmaceutically active substances is known in the art.Except insofar as any conventional media or agent is incompatible withthe active humanized antibody, use thereof in the pharmaceuticalcompositions of the present invention is contemplated.

Proper fluidity may be maintained, for example, by the use of coatingmaterials, such as lecithin, by the maintenance of the required particlesize in the case of dispersions, and by the use of surfactants.

Pharmaceutical compositions of the present invention may also comprisepharmaceutically acceptable antioxidants for instance (1) water solubleantioxidants, such as ascorbic acid, cysteine hydrochloride, sodiumbisulfate, sodium metabisulfite, sodium sulfite and the like; (2)oil-soluble antioxidants, such as ascorbyl palmitate, butylatedhydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propylgallate, alpha-tocopherol, and the like; and (3) metal chelating agents,such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol,tartaric acid, phosphoric acid, and the like.

Pharmaceutical compositions of the present invention may also compriseisotonicity agents, such as sugars, polyalcohols, such as mannitol,sorbitol, glycerol or sodium chloride in the compositions.

The pharmaceutical compositions of the present invention may alsocontain one or more adjuvants appropriate for the chosen route ofadministration such as preservatives, wetting agents, emulsifyingagents, dispersing agents, preservatives or buffers, which may enhancethe shelf life or effectiveness of the pharmaceutical composition. Thehumanized antibodies of the present invention may be prepared withcarriers that will protect the humanized antibody against rapid release,such as a controlled release formulation, including implants,transdermal patches, and microencapsulated delivery systems. Suchcarriers may include gelatin, glyceryl monostearate, glyceryldistearate, biodegradable, biocompatible polymers such as ethylene vinylacetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters,and polylactic acid alone or with a wax, or other materials well knownin the art. Methods for the preparation of such formulations aregenerally known to those skilled in the art. See, e.g., Sustained andControlled Release Drug Delivery Systems, J. R. Robinson, ed., MarcelDekker, Inc., New York, 1978.

In one embodiment, the antibodies of the present invention may beformulated to ensure proper distribution in vivo. Pharmaceuticallyacceptable carriers for parenteral administration include sterileaqueous solutions or dispersions and sterile powders for theextemporaneous preparation of sterile injectable solutions ordispersion. The use of such media and agents for pharmaceutically activesubstances is known in the art. Except insofar as any conventional mediaor agent is incompatible with the humanized antibody, use thereof in thepharmaceutical compositions of the present invention is contemplated.

Pharmaceutical compositions for injection must typically be sterile andstable under the conditions of manufacture and storage. The compositionmay be formulated as a solution, microemulsion, liposome, or otherordered structure suitable to high drug concentration. The carrier maybe a aqueous or non-aqueous solvent or dispersion medium containing forinstance water, ethanol, polyols (such as glycerol, propylene glycol,polyethylene glycol, and the like), and suitable mixtures thereof,vegetable oils, such as olive oil, and injectable organic esters, suchas ethyl oleate. The proper fluidity may be maintained, for example, bythe use of a coating such as lecithin, by the maintenance of therequired particle size in the case of dispersion and by the use ofsurfactants. In many cases, it will be preferable to include isotonicagents, for example, sugars, polyalcohols such as glycerol, mannitol,sorbitol, or sodium chloride in the composition. Prolonged absorption ofthe injectable compositions may be brought about by including in thecomposition an agent that delays absorption, for example, monostearatesalts and gelatin. Sterile injectable solutions may be prepared byincorporating the humanized antibody in the required amount in anappropriate solvent with one or a combination of ingredients e.g. asenumerated above, as required, followed by sterilizationmicrofiltration. Generally, dispersions are prepared by incorporatingthe humanized antibody into a sterile vehicle that contains a basicdispersion medium and the required other ingredients e.g. from thoseenumerated above. In the case of sterile powders for the preparation ofsterile injectable solutions, examples of methods of preparation arevacuum drying and freeze-drying (lyophilization) that yield a powder ofthe active ingredient plus any additional desired ingredient from apreviously sterile-filtered solution thereof.

Sterile injectable solutions may be prepared by incorporating thehumanized antibody in the required amount in an appropriate solvent withone or a combination of ingredients enumerated above, as required,followed by sterilization microfiltration. Generally, dispersions areprepared by incorporating the humanized antibody into a sterile vehiclethat contains a basic dispersion medium and the required otheringredients from those enumerated above. In the case of sterile powdersfor the preparation of sterile injectable solutions, examples of methodsof preparation are vacuum drying and freeze-drying (lyophilization) thatyield a powder of the active ingredient plus any additional desiredingredient from a previously sterile-filtered solution thereof.

Dosage regimens in the above methods of treatment and uses are adjustedto provide the optimum desired response (e.g., a therapeutic response).For example, a single bolus may be administered, several divided dosesmay be administered over time or the dose may be proportionally reducedor increased as indicated by the exigencies of the therapeuticsituation. Parenteral compositions may be formulated in dosage unit formfor ease of administration and uniformity of dosage. Dosage unit form asused herein refers to physically discrete units suited as unitarydosages for the subjects to be treated; each unit contains apredetermined quantity of humanized antibody calculated to produce thedesired therapeutic effect in association with the requiredpharmaceutical carrier. The specification for the dosage unit forms ofthe present invention are dictated by and directly dependent on (a) theunique characteristics of the humanized antibody and the particulartherapeutic effect to be achieved, and (b) the limitations inherent inthe art of compounding such an humanized antibody for the treatment ofsensitivity in individuals.

The effective dosages and the dosage regimens for the humanizedantibodies of the invention depend on the disease or condition to betreated and may be determined by the persons skilled in the art. Anexemplary, non-limiting range for a therapeutically effective amount ofan antibody of the present invention is about 0.1-10 mg/kg/body weight,such as about 0.1-5 mg/kg/body weight, for example about 0.1-2mg/kg/body weight, such as about 0.1-1 mg/kg/body weight, for instanceabout 0.15, about 0.2, about 0.5, about 1, about 1.5 or about 2mg/kg/body weight.

A physician or veterinarian having ordinary skill in the art may readilydetermine and prescribe the effective amount of the pharmaceuticalcomposition required. For example, the physician or veterinarian couldstart doses of the specified humanized antibody employed in thepharmaceutical composition at levels lower than that required in orderto achieve the desired therapeutic effect and gradually increase thedosage until the desired effect is achieved. In general, a suitabledaily dose of a composition of the present invention will be that amountof the humanized antibody which is the lowest dose effective to producea therapeutic effect. Such an effective dose will generally depend uponthe factors described above. Administration may e.g. be intravenous,intramuscular, intraperitoneal, or subcutaneous, and for instanceadministered proximal to the site of the target. If desired, theeffective daily dose of a pharmaceutical composition may be administeredas two, three, four, five, six or more sub-doses administered separatelyat appropriate intervals throughout the day, optionally, in unit dosageforms. While it is possible for a humanized antibody of the presentinvention to be administered alone, it is preferable to administer thehumanized antibody as a pharmaceutical composition as described above.

Examples 1. Humanization Approach to Generate N3pE-Aβ Specific HumanizedAntibodies

N3pE-Aβ specific mouse monoclonal antibody clones #6, #17 and #24 wereobtained from hybridoma cell lines 6-1-6, 17-4-3, and 24-2-3, which havebeen deposited in accordance with the Budapest Treaty and are availableat the Deutsche Sammlung für Mikroorganismen und Zellkulturen (DSMZ) inBraunschweig, DE, with a deposit date of Jun. 17, 2008, and with therespective deposit numbers

-   -   (clone 6-1-6): DSM ACC2924    -   (clone 17-4-3): DSM ACC2925    -   (clone 24-2-3): DSM ACC2926.

The first step in humanization process of antibody clones #6, #17 and#24 was the definition of the CDRs in the variable domains of light andheavy chains. By Rosetta Antibody Modeling Server(http://antibody.graylab.jhu.edu) the CDRs were predicted. FIG. 1 showsexemplary the CDRs predicted for clone #6.

To select the appropriate framework for CDR-Grafting human sequenceswith the highest similarity to the non-human antibody need to beidentified. By Blast analysis the variable domain of the light chain(Lv) and the heavy chain (Hv) were separately fitted with the pool ofpublished human sequences. For the light chain a human antibody sequencewith 82% identity was found, which belongs to the class of kappa LC. Thehighest homology for the heavy chain has a human amino acid sequencewith 62% identity.

To group the selected human antibody framework sequence to germline genesequences, a Blast search was performed in the germline libary IMGT. Forlight chain clone#6 and clone#24 a sequence with the sequence codeIGKV2-30*01 was found. The light chain variable region of clone#17 wasmost similar to IGKV2-30*02. The heavy chain variable region codes couldbe identified with IGHV1-3*01, IGHV1-69*13 and IGHV3-48*01 correspondingto clone#6, clone#17 and clone#24, respectively. Table 1 shows theframework parameters of variable regions of the humanized antibodies.

TABLE 1 Framework parameters of variable regions of the humanizedantibodies. Lv Hv clone# Aa acc nr. family aa acc nr. family 6BAC01730.1 AB064102.1 IGKV2- AAS85817.1 AY392875.1. IGHV1-3*01 30*01 24BAC01730.1 AB064102.1 IGKV2- AAD30405.1 AF115119.1 IGHV1- 30*01 69*13 17BAC01734.1 AB064106.1 IGKV2- AAS858165 AY392925.1 IGHV3- 30*02 48*01 aa= amino acid sequence, acc nr. = accession number, family = gene family

By CDR grafting the CDRs of the mouse antibody clones #6, #17 and #24were combined with the respective human antibody framework to create ahumanized antibody. The heavy chain constant region of human IgG1 wasused for reconstitution the entire antibodies. The light chain variabledomains were fused to the human kappa chain constant region.

2. RNA Isolation and cDNA Synthesis

As source for constant sequences, RNA of human B cells was isolated bylysis of 500 μl whole blood with 5 ml 1×FACS Lysis Solution (BectonDickinson) for 10 minutes at room temperature. The lysate wascentrifuged at 300 g for 5 min; the pellet was washed two times with PBSand was then resolved in 350 μl RA1 Buffer of Nucleo Spin® RNA II(Macherey-Nagel) and added with 3.5 μl 0.5M TCEP (SIGMA). The RNA wasisolated by manufacturers' instructions. 10 μl of RNA was firstincubated with 1 μl 0.5 μg/μl OligodT Primer (Invitrogen) and 1 μl 10 mMdNTPs for 5 min at 65° C. Then 4 μl of 5× First Strand Buffer(Invitrogen), 2 μl of 100 mM DTT and 0.5 μl SuperScript III ReverseTranscriptase (Invitrogen) was added to 20 μl and mix was incubated for5 minutes at 25° C., 50 min at 50° C. and 15 min at 70° C. By PCR withprimer pairs shown in Table 2, synthesized cDNA of constant region oflight and heavy chain could be amplified.

TABLE 2 Primer for cloning of constant region SEQ ID NO Name Sequence 44hkappa5′ ACTGTGGCTGCACCATCTGTCTTC 45 hkappa3′ CTAACACTCTCCCCTGTTGAAGCTC46 hIgG1Hc5′1 AGGGAACCCTGGTCACCGTCTCC 47 hIgG1Hc3′TCATTTACCCGGAGACAGGGAGAGG

For amplification of the PCR product of humanized light chain clone#6following forward and reverse primers were used:

RT_chim_humKI6f: (SEQ ID NO: 48) CAAGTCAGAGCCTCTTATATAGTG;RT_chim_humKI6r: (SEQ ID NO: 49) GTACCTTGCACGCAGTAATAAAC.

For amplification of the reference gene mouse HPRT Primer were used.

To perform the amplification 7.5 μl Sybergreen (Firma), 1 μl Primerforward (25 pmol/μl), 1 μl Primer reverse (25 pmol/μl), 5.5 μl ddH₂O and1 μl cDNA were used in cycler.

3. Expression of Recombinant Antibody in CHO Cells by Separately CloningLC and HC into Two Different Expression Plasmids

The sequences of the light and heavy chain of the humanized antibodieswere separately cloned into two different mammalian expression vectors,pCDNA3.1 and HC-pOptiVEC respectively. To identify the optimalcombination of vectors to express the recombinant antibody in CHO cellculture, different plasmid combinations were used to perform transientexpressions in adherent CHO cells. In a second step, it was investigatedwhether different DNA ratios between LC and HC plasmid influences theexpression level. With transfection of 3 μg LC-pCDNA3.1 and 1 μgHC-pOptiVEC, an increased expression level was found.

For further adherent CHO cells expression of humanized antibody, plasmidcombination of LC-pCDNA3.1 and 1 μg HC-pOptiVEC and a plasmid DNA ratioof LC 3:1 HC was used.

Freestyle™ CHO suspension cells were used in the following transfectionsto cultivate a higher amount of transient expressing cells which togenerate recombinant antibodies. First was tested whether an excess ofLC plasmid could improve the expression of antibody like in case of theadherent cells. Like in adherent CHO cells a LC to HC plasmid DNA ratioof 1:1 and 3.1 was used. Western blot analysis revealed that an excessof LC plasmid increases the expression of humanized antibody as in thecase of adherent CHO cells. By measurement of cell viability it becomeobvious, that the cell viability decreases to about 50% of transfectedcells after 6 days. After day six, no further increase of antibody levelin supernatant was detectable. Consequently, culture supernatants wereharvest at day six in the case of following transfections.

To investigate if the produced antibodies are efficient transported intothe cell supernatant, a cell lysate sample was applied to SDS PAGE,analyzed by Western blot. GAPDH, a housekeeping cytoplasmic protein wasused for reference loading comparable amounts of cell lysate protein tothe SDS gel. In cell lysate of humanized antibody expressing CHO cells astrong band of 120 kDa occurs migrating at the same size as detected inthe cell supernatant.

4. Purification of Recombinant Antibody by Protein G Chromatography

The humanized antibody clone#6 was purified to investigate the antigenbinding property of the protein in comparison with the original murineantibody. Therefore 300 ml supernatant with expressed chimeric andhumanized antibody was produced and purified by Protein G chromatography(FIG. 2). Because the amount of expressed antibody was very low, theyield was less than 0.1 μg/ml, in total 25 μg purified protein. Theeluted antibody was concentrated to about 200 μg/ml and 2 μg protein wasapplied to SDS-PAGE following coomassie staining (FIG. 2).

5. Surface Plasmon Resonance Measurement to Compare Antigen Binding ofMurine and Humanized Antibody

Surface Plasmon Resonance measurement was used to investigate thebinding efficacy of humanized antibody to AβpE3-18 (FIG. 3). To preventmass transfer and avidity effects during measurement, the followingprocedure was used.

First a polyclonal α-human antibody was coupled to an SPR-Chipsubsequent loaded with the humanized antibody until the Response Unitwas more than 1000.

Kinetic measurement was performed at different concentrations (of 5 to1000 nM) of AβpE3-18-peptide. The graphs of the measured series areshown as an overlay plot with the sensorgrams, corrected by thesensogram measuring the running buffer, aligned at the time of injectionand the baseline adjusted to zero before injection. The results areevaluated according to a simple 1:1 interaction model (Langmuir fit),which promote the k_(off) and k_(on) rate constants. In FIG. 3 theSPR-binding curves are shown, consisting of association and dissociationcurve.

Apparent kinetic constants according to the 1:1 Langmuir fitting arelisted in Table 3. Due to the fact that the humanized antibody isnon-covalently bound to the chip surface, small amounts of antibodymolecules were washed out during the measurement. Therefore the Rmaxvalues were fitted locally for every single sensogram.

TABLE 3 Statistics of Langmuir fit in kinetics of humanized antibodyclone#6 Aβ_(pE3-18) in k_(on) k_(off) Rmax Req nM (1/Ms) (1/s) (RU) RI(RU) KA (1/M) KD (M) (RU) Chi2 8.09E+05 0.0222 0.303 3.65E+07 2.74E−080.803 0 1.19 0 1. measurement 5 18.1 2.79 10 15.5 4.15 20 16.7 7.05 5016.9 10.9 100 18.7 14.7 200 20.2 17.7 500 21.7 20.5 1000 22.7 22.1 0 0 02. measurement 5 11.4 1.76 10 15.5 4.14 20 17.5 7.39 50 16.9 10.9 10018.3 14.4 200 20.2 17.7 500 21 19.9 1000 22.3 21.7

The plot of the curves during association of peptide (FIG. 3A, upperinset) fits very well, whereas the plot of peptide dissociation curves(FIG. 3A, lower inset) don't localize accurate on experimental curves.With the fit the kinetic parameter k_(off) of 22.2 10⁻³ 1/s was computed(Table 2). This parameter describes the time which is necessary forremoving the half of bounded peptide. This means that during 1/0.0222s=45 s the half of injected peptide is removed (see FIG. 3A, lowerinset). Because Langmuir fit doesn't match very well during dissociationphase, K_(D) determination was performed by fitting R_(equ) values overpeptide concentration (FIG. 3B) using following equation:R_(equ)=R_(max)·K_(A)·c/(1+K_(A)·c), at which R_(equ) the signal inequilibrium with corresponding concentration c are variables and R_(max)and K_(A) the constants to fit. K_(D) can be calculated by 1/K_(A).

By structural analysis of mouse Fab fragment antibody clone#6 wasconcluded that Thr97 in HC could have an important effect on bindingaffinity to AβpE3. After replacement of Ala97 by Thr97 in the humanizedantibody clone#6 an improved binding affinity was yielded with nearlythe same K_(D) value compared to the mouse antibody clone#6. Higheramount of expressed antibody was required to prove these findings byadditional ITC measurement.

Therefore the expression level was increased by a replacement of LC andHC signal sequences of humanized antibody T97 variant by the murinesequences. In a second step generation of a stable cell line wasperformed to increase antibody production.

Table 4 shows the apparent kinetic constants according to the 1:1Langmuir fitting of further sequence variants of humanized antibodyclone #6:

K_(D) kon koff Variant (nM) (s⁻¹M⁻¹) (s⁻¹) VH: SEQ ID NO: 24 9.36 6.16 ×10⁵ 5.76 × 10⁻³ VL: SEQ ID NO: 14 VH: SEQ ID NO: 27 5.09 6.35 × 10⁵ 3.23× 10⁻³ VL: SEQ ID NO: 14 VH: SEQ ID NO: 27 15.5 2.67 × 10⁵ 4.15 × 10⁻³VL: SEQ ID NO: 15 VH: SEQ ID NO: 27 7.35 5.62 × 10⁵ 4.13 × 10⁻³ VL: SEQID NO: 13 VH: SEQ ID NO: 70 8.43 4.23 × 10⁵ 3.57 × 10⁻³ VL: SEQ ID NO:14

6. Stable Cell Line Generation of Humanized Antibody Variant HC T97

For stable cell line development first a pool of stable CHO-DG44 celllines were generated by treatment with different concentrations ofmethotrexate (MTX). The best expression was detected accompanied by alow amount of dying cells at a concentration of 0.5 μM MTX (FIG. 14A,lane 2). Thus, cells with pretreatment of 0.5 μM MTX were used forclonal selection by limiting dilution.

One hundred potential antibody expressing clones were found after clonalselection. The supernatant of these clones were diluted 1:20 in HBS-EPbuffer and analyzed by SPR using an AβpE3-18 coupled chip. A calibrationcurve was used to determine the antibody concentration in supernatant.18 clones were separated which show a started antibody concentrationover 0.15 μg/ml after 3 days of expression. These clones were scaled upin 24 well format and supernatants were collected and analyzed byWestern blot (FIG. 4B). Five of these clones revealed good expressionand were further scaled up until 30 ml shaking culture. After 7 dayexpression time the expression level was measured by SPR (FIG. 4C).Clone 9A has shown the highest antibody concentration after 7 daycultivation. Therefore this clone was used for expression higher amountsof supernatant. The cultivation was performed without further selectionpressure to get higher cell viability. Three liters of antibodycontaining supernatant were collected.

7. Purification of Humanized Antibody Clone#6 by Protein GChromatography

One liter of supernatant was diluted with one liter 40 mM Na₂HPO₄ pH 7and applied overnight to 5 ml Protein G column at 4° C. After thatcolumn was washed with binding buffer (20 mM Na₂HPO₄, pH 7, fractionA1-Aβ) and then with high-salt buffer (2 M NaCl, 40 mM Na₂HPO₄ pH 7,fraction A4-A7) to remove unspecific bound proteins. The antibody waseluted with 0.1M Glycin-HCl, pH 2.7 from column and immediatelyneutralized by 1 M Tris pH 9. Fractions were collected and 24 μl wereloaded onto 12% SDS PAGE, respectively. The fractions A12+B1 were pooledand used for K_(D) determination by ITC. Overall, 2.5 mg antibody waspurified from one liter culture supernatant.

8. ITC Measurement of Humanized Antibody Clone#6 with AβpE3-18

For determination of the K_(D)-Value, the humanized antibody clone#6 wasdiluted to a concentration of 1 μM. The ligand AβpE3-18 with aconcentration of 20 μM was titrated at 293.15K. After integration of theraw data of ITC-measurement (FIG. 5B, bottom) the stoichiometry of 1.5was calculated. The calculated K_(D)-Value was 2.7 nM according quitewell with value of 5.3 nM calculated from the SPR data characterizingthe humanized clone#6 HC T97 ligand binding in solvent as a high affineinteraction. The interaction is driven mainly by enthalpic contributions(ΔH=−23.45 kcal/mol) and opposed by entropic penalties (TΔS=−11.96kcal/mol), which is typical for structural rearrangement at the bindingsite by formation of hydrogen bonds yielding in significant loss ofdegrees of freedom.

9. Humanization, Expression and Purification of the Two FurtherAβpE3-Specific Antibodies Clone#17 and Clone#24

The humanization, expression and purification of the two furtherAβpE3-specific antibodies clone#17 and clone#24 was performed using thesame protocols, materials and methods as well as experimental conditionsas used for clone#6.

10. SPR Measurement with Humanized Antibody Clone#24 and Clone#17

SPR measurements using the pE3-Aβ18 ligand revealed that both humanizedantibodies are capable to bind the pE3-Aβ peptide. The rate constantsand the K_(D) value could be calculated very well by Langmuir 1:1 modelfit. The origin K_(D) values of mouse antibodies to pE3-Aβ are shown intable 5. It became obvious that the k_(off) value of humanized antibodyclone#24 is 20 fold higher than the k_(off) value of mouse antibodyclone#24. This means that after humanization the pE3-Aβ peptidedissociates faster from the antigen binding pocket of the antibody.

TABLE 5 Determination of k_(on), k_(off) and K_(D) value of differentAβ peptides Abeta sequence Ab clone# KD in nM k_(on) in 1/Ms k_(off) in1/s human pEFRHDSGYEVHHQKLV mouse 6 6.7 6.89E+05 4.58E−03 pE3-18 (SEQ IDNO: 50) mouse 24 2.2 1.76E+05 0.38E−03 mouse 17 1.8 19.5E+05 3.46E−03human 6 5.3 6.89E+05 4.58E−03 human 24 16 4.91E+05 7.84E−03 human 17 1.519.9E+05 2.90E−03 human DAEFRHDSGYEVHHQKLV mouse 6 — — — 1-18 (SEQ IDNO: 51) mouse 24 — — — mouse 17 — — — human 6 — — — human 17 — — — humanAEFRHDSGYEVHHQKLV mouse 6 n.d. — — 2-18 (SEQ ID NO: 52) mouse 24 n.d. —— mouse 17 490 0.0741E+05   3.63E−03 human 6 n.d. — — human 17 1900.149E+05  2.84E−03 human FRHDSGYEVHHQKLV mouse 6 — — — 4-18 (SEQ ID NO:53) mouse 24 — — — mouse 17 — — — human 6 — — — human 17 — — —

11. Binding to Fc Gamma Receptors

The binding of two antibodies, which either comprised the human IgG1 Fcwild-type region of SEQ ID NO: 73 or the K324A mutant variant thereof(SEQ ID NO: 74), to different Fc gamma receptors (CD16A, CD32A, CD32B,and CD64) was compared.

The K324A mutant was produced by site-directed mutagenesis. The bindingwas measured in a FACS based bioassay to Chinese Hamster Ovary (CHO)cells stably expressing full length human CD16A, CD32A, CD32B, or CD64.Both antibodies were incubated with each cell line at 7 differentconcentrations for one hour followed by washing. Receptor-bound H6 orH67 was detected with fluorochrome conjugated-anti-Fab′. Bindingcapacity was measured by FACS and the Kd and Bmax were calculated bynon-linear regression.

Results: Both antibodies showed comparable binding to all receptors. SeeFIGS. 6, 7, 8 and 9.

12. Binding to C1q

The binding of two antibodies, which either comprised the human IgG1 Fcwild-type region of SEQ ID NO: 73 or the K324A mutant variant thereof(SEQ ID NO: 74), to C1q was compared in order to better characterize theeffector functions of the antibodies.

A number of assay formats of binding of the antibodies to C1q wastested, including

a) direct binding of the two antibodies to the plate and then biding toC1q in solution; andb) streptavidin coated plates first incubated with biotinylated pE-Aβpeptide, binding to antibodies and then C1q.

In summary, format a) produced best results. The procedure is summarizedbelow:

The ELISA plate was coated with the antibody, comprising the human IgG1Fc wild-type region of SEQ ID NO: 73, the K324A mutant variant thereof(SEQ ID NO: 74), and a K324A control (hu14.18K324A) that does not bindC1q at 10, 8, 6, 4, 3, 2, 1 and 0 μg/ml in triplicate and incubated at4° C. overnight. Next day, the plate was washed three times with 1×PBSand then blocked with 1% BSA in 1×PBS at 50 μl/well. C1q (Sigma, Cat. #C1740) was added to each well at 2 μg/ml in blocking buffer andincubated for 1 hour at room temperature. The plate was then washedthree times with 200 μl of 1×PBS. Anti-C1q-HRP (Thermo, Cat. #PA1-84324)was added to the plate to detect the binding at a 1:250 dilution inblocking buffer (50 μl/well) for 1 hour. The plate was washed againthree times with 200 μl of 1×PBS. 50 μl of TMB (Invitrogen, Cat.#002023) was added to each well to visualize the interaction(Invitrogen, Cat. #002023) for 2 min. 50 μl of stop solution ((1MSulfuric Acid) was added to each well before reading the absorbance at450 nm.

Results: The antibody, which comprised the human IgG1 Fc wild-typeregion of SEQ ID NO: 73 did bind to C1q. The K324A mutant variantthereof (comprising the IgG1 Fc region of SEQ ID NO: 74), did not bindto C1q. See also FIG. 10.

13. Immunohistochemistry

With IHC the antigen Aβ N3pE can be localized in cerebral tissuesections. Therefore the humanized antibodies of the invention were usedfor detection of Aβ N3pE.

For the IHC human cerebral tissue sections of the hippocampus and thefrontal cortex from AD patients and furthermore cerebral tissue sectionsof hippocampus from existing animal models for Alzheimer's disease asdescribed herein can be used. These mouse models show increased brain Aβlevels followed by development of neuritic plaques. The tissue sectionswere paraffin-embedded and serial cut. The sections were stained withhematoxylin to colored nuclei of cells and then immunostained with theanti Aβ N3pE antibodies of the invention. The tissue section preparationand staining were performed in accordance with standard methodology.

14. Treatment of Alzheimer Mice In Vivo

A total of 62 male mice were utilized in this study. Prior to the startof immunization, four mice of an existing mouse model for Alzheimer'sdisease (avg. 5.6 mo±0.45) mice were sacrificed as baseline controls toassess cerebral Aβ plaque burden at the commencement of treatment. Theremaining mice were divided into four groups and received the followingtreatment: 250 μl sterile PBS (n=12; avg. 5.89 mo±0.13), 200 μg of ahumanized antibody of the invention. A group of age- and gender-matchedWt littermates were injected with 250 μl PBS (n=12; avg. 5.80 mo±0.12)and served as behavioral controls. Mice were treated with a total volumeof 250 μl (antibody or PBS) via intraperitoneal injection for 28 weeks.

Euthanasia and Tissue Preparation

Mice were euthanized, perfused and plasma harvested at 6 months(baseline) or 13 months of age. The brain was extracted and dividedsagittally. The hippocampus, cortex and cerebellum were dissected fromone hemisphere and snap frozen for biochemical analyses. The otherhemisphere was drop-fixed in 4% parafomaldehyde (Electron MicroscopySciences) for 24 h at 4° C., cryoprotected in graded sucrose solutionsat 4° C. and embedded in OCT compound (Tissue Tek).

1. A humanized antibody or a functional variant thereof, wherein thevariable part of the light chain of said antibody comprises an aminoacid sequence selected from the group consisting of SEQ ID NO: 7, SEQ IDNO: 28, and SEQ ID NO:
 36. 2. A humanized antibody or a functionalvariant thereof, wherein the variable part of the light chain of saidantibody comprises an amino acid sequence selected from the groupconsisting of SEQ ID NO: 17, SEQ ID NO: 32, and SEQ ID NO:
 40. 3. Thehumanized antibody of claim 1, comprising the CDR regions: V_(L) CDR1:SEQ ID NO: 8; V_(L) CDR2: SEQ ID NO: 9; and V_(L) CDR3: SEQ ID NO: 10 inthe light chain.
 4. The humanized antibody of claim 1, wherein thevariable part of the light chain comprises the amino acid sequence ofSEQ ID NO:
 11. 5. The humanized antibody of claim 4, comprising the CDRregions: V_(L) CDR1: SEQ ID NO: 12, V_(L) CDR2: SEQ ID NO: 9; and V_(L)CDR3: SEQ ID NO: 10 in the light chain.
 6. The humanized antibody ofclaim 1, wherein the variable part of the light chain comprises theamino acid sequence of SEQ ID NO:
 13. 7. The humanized antibody of claim6, comprising the CDR regions V_(L) CDR1 of SEQ ID NO: 12, V_(L) CDR2SEQ ID NO: 9 and V_(L) CDR3 of SEQ ID NO: 10 in the light chain.
 8. Thehumanized antibody of claim 1, wherein the variable part of the lightchain comprises the amino acid sequence of SEQ ID NO:
 14. 9. Thehumanized antibody of claim 8, comprising the CDR regions V_(L) CDR1 ofSEQ ID NO: 12, V_(L) CDR2 of SEQ ID NO: 9 and V_(L) CDR3 of SEQ ID NO:10 in the light chain.
 10. The humanized antibody of claim 1, whereinthe variable part of the light chain comprises the amino acid sequenceof SEQ ID NO:
 15. 11. The humanized antibody of claim 10, comprising theCDR regions: V_(L) CDR1: SEQ ID NO: 16, V_(L) CDR2: SEQ ID NO: 9; andV_(L) CDR3: SEQ ID NO: 10 in the light chain.
 12. The humanized antibodyaccording to claim 1, comprising the CDR regions: V_(H) CDR1: SEQ ID NO:18, V_(H) CDR2: SEQ ID NO: 19; V_(H) CDR3: SEQ ID NO: 20 in the heavychain.
 13. The humanized antibody of claim 12, wherein the variable partof the heavy chain comprises the amino acid sequence of SEQ ID NO: 21.14. The humanized antibody of claim 13, comprising the CDR regions:V_(H) CDR1: SEQ ID NO: 22, V_(H) CDR2: SEQ ID NO: 23; V_(H) CDR3: SEQ IDNO: 20 in the heavy chain.
 15. The humanized antibody of claim 12,wherein the variable part of the heavy chain comprises the amino acidsequence of SEQ ID NO:
 24. 16. The humanized antibody of claim 15,comprising the CDR regions: V_(H) CDR1: SEQ ID NO: 25, V_(H) CDR2: SEQID NO: 26; V_(H) CDR3: SEQ ID NO: 20 in the heavy chain.
 17. Thehumanized antibody of claim 12, wherein the variable part of the heavychain comprises the amino acid sequence of SEQ ID NO:
 27. 18. Thehumanized antibody of claim 17, comprising the CDR regions: V_(H) CDR1:SEQ ID NO: 25, V_(H) CDR2: SEQ ID NO: 26; V_(H) CDR3: SEQ ID NO: 20 inthe heavy chain.
 19. The humanized antibody of claim 12, wherein thevariable part of the heavy chain comprises the amino acid sequence ofSEQ ID NO:
 66. 20. The humanized antibody of claim 19, comprising theCDR regions: V_(H) CDR1: SEQ ID NO: 25, V_(H) CDR2: SEQ ID NO: 67; andV_(H) CDR3: SEQ ID NO: 20 in the heavy chain.
 21. The humanized antibodyof claim 12, wherein the variable part of the heavy chain comprises theamino acid sequence of SEQ ID NO:
 68. 22. The humanized antibody ofclaim 19, comprising the CDR regions: V_(H) CDR1: SEQ ID NO: 25, V_(H)CDR2: SEQ ID NO: 69; and V_(H) CDR3: SEQ ID NO: 20 in the heavy chain.23. The humanized antibody of claim 12, wherein the variable part of theheavy chain comprises the amino acid sequence of SEQ ID NO:
 70. 24. Thehumanized antibody of claim 22, comprising the CDR regions: V_(H) CDR1:SEQ ID NO: 25, V_(H) CDR2: SEQ ID NO: 71; and V_(H) CDR3: SEQ ID NO: 20in the heavy chain.
 25. The humanized antibody of claim 1, wherein thevariable part of the light chain of said antibody comprises the aminoacid sequence of SEQ ID NO:
 28. 26. The humanized antibody of claim 25,comprising the CDR regions: V_(L) CDR1: SEQ ID NO: 29, V_(L) CDR2: SEQID NO: 30; and V_(L) CDR3: SEQ ID NO: 31 in the light chain.
 27. Thehumanized antibody of claim 26, wherein the variable part of the heavychain of said antibody comprises the amino acid sequence of SEQ ID NO:32.
 28. The humanized antibody of claim 27, comprising the CDR regions:V_(H) CDR1: SEQ ID NO: 33, V_(H) CDR2: SEQ ID NO: 34; V_(H) CDR3: SEQ IDNO: 35 in the heavy chain.
 29. The humanized antibody of claim 1,wherein the variable part of the light chain of said antibody comprisesthe amino acid sequence of SEQ ID NO:
 36. 30. The humanized antibody ofclaim 29, comprising the CDR regions: V_(L) CDR1: SEQ ID NO: 37, V_(L)CDR2: SEQ ID NO: 38; and V_(L) CDR3: SEQ ID NO: 39 in the light chain.31. The humanized antibody of claim 30, wherein the variable part of theheavy chain of said antibody comprises the amino acid sequence of SEQ IDNO:
 40. 32. The humanized antibody of claim 31, comprising the CDRregions: V_(H) CDR1: SEQ ID NO: 41, V_(H) CDR2: SEQ ID NO: 42; V_(H)CDR3: SEQ ID NO: 43 in the heavy chain.
 33. The humanized antibodyaccording to claim 1, having a human IgG1 Fc region which comprises anamino acid sequence selected from the group consisting of SEQ ID NO: 73and SEQ ID NO:
 74. 34. The humanized antibody according to claim 1,having a human IgG1 Fc region which comprises the amino acid sequence ofSEQ ID NO:
 74. 35. The humanized antibody according to claim 1, whereinthe variable part of the light chain comprises the amino acid sequenceof SEQ ID NO: 14; and having a variable part of a heavy chain whichcomprises the amino acid sequence of SEQ ID NO: 27; and having a humanIgG1 Fc region which comprises the amino acid sequence of SEQ ID NO: 74.36. The humanized antibody according to claim 1, having a variable partof a light chain, a variable part of a heavy chain, and a human IgG1 Fcregion, wherein: the variable part of the light chain comprises theamino acid sequence of SEQ ID NO: 14; and the variable part of the heavychain comprises the amino acid sequence of SEQ ID NO: 27; and the humanIgG1 Fc region comprises the amino acid sequence of SEQ ID NO: 74; thevariable part of the light chain comprises the CDR regions V_(L) CDR1 ofSEQ ID NO: 12, V_(L) CDR2 of SEQ ID NO: 9 and V_(L) CDR3 of SEQ ID NO:10; and the variable part of the heavy chain comprises the CDR regionsV_(H) CDR1 of SEQ ID NO: 25, V_(H) CDR2 of SEQ ID NO: 26 and V_(H) CDR3of SEQ ID NO:
 20. 37. The humanized antibody according to claim 1,having a variable part of a light chain, a variable part of a heavychain, and a human IgG1 Fc region, wherein the variable part of thelight chain comprises the amino acid sequence of SEQ ID NO: 14; and thevariable part of the heavy chain comprises the amino acid sequence ofSEQ ID NO: 70; and the human IgG1 Fc region comprises the amino acidsequence of SEQ ID NO:
 74. 38. The humanized antibody according to claim1, having a variable part of a light chain, a variable part of a heavychain, and a human IgG1 Fc region, wherein: the variable part of thelight chain comprises the amino acid sequence of SEQ ID NO: 14; and thevariable part of the heavy chain comprises the amino acid sequence ofSEQ ID NO: 70; and the human IgG1 Fc region comprises the amino acidsequence of SEQ ID NO: 74; the variable part of the light chaincomprises the CDR regions V_(L) CDR1 of SEQ ID NO: 12, V_(L) CDR2 of SEQID NO: 9 and V_(L) CDR3 of SEQ ID NO: 10; and the variable part of theheavy chain comprises the CDR regions V_(H) CDR1 of SEQ ID NO: 25, V_(H)CDR2 of SEQ ID NO: 71 and V_(H) CDR3 of SEQ ID NO:
 20. 39. The humanizedantibody according to claim 1, wherein said humanized antibodyspecifically binds to a pyroglutamate carrying N-terminus of the Aβ N3pEepitope.
 40. The humanized antibody according to claim 1, wherein saidhumanized antibody binds specifically to an epitope selected from thegroup consisting of SEQ ID NO: 50, SEQ ID NO: 54, SEQ ID NO: 55, SEQ IDNO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60, SEQID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 72 SEQ ID NO: 64,and SEQ ID NO:
 65. 41. The humanized antibody according to claim 1,wherein said humanized antibody binds to an Aβ N3pE variant, wherein theAβ N3pE variant is pE-Aβ_(3-x), and wherein x is an integer having avalue in the range of 19 to
 42. 42. The humanized antibody of claim 41,wherein the Aβ N3pE variant is selected from: pE-Aβ₃₋₃₈, pE-Aβ₃₋₄₀, andpE-Aβ₃₋₄₂.
 43. The humanized antibody according to claim 1, wherein saidhumanized antibody does not bind to epitopes that do not carry apyroglutamate at the N-terminus.
 44. A pharmaceutical compositioncomprising the humanized antibody according to claim
 1. 45. Thepharmaceutical composition of claim 44, further comprising a furtherbiologically active substance and/or a pharmaceutically acceptablecarrier and/or a diluent and/or an excipient.
 46. The pharmaceuticalcomposition of claim 45, wherein said further biologically activesubstance is selected from a neutron-transmission enhancer,psychotherapeutic drug, an acetylcholine esterase inhibitor, acalcium-channel blocker, a biogenic amine, a benzodiazepinetranquilizer, an acetylcholine synthesis, storage or release enhancer,an acetylcholine postsynaptic receptor agonist, a monoamine oxidase-A or-B inhibitor, a N-methyl-D-aspartate glutamate receptor antagonist, anon-steroidal anti-inflammatory drug, an antioxidant, and a serotonergicreceptor antagonist.
 47. The pharmaceutical composition of claim 45,wherein said further biologically active substance is selected from thegroup consisting of a compound effective against oxidative stress, ananti-apoptotic compound, a metal chelator, an inhibitor of DNA repair,an α-secretase activator, a β- and γ-secretase inhibitor, a tau protein,a neurotransmitter, a β-sheet breaker, an attractant for amyloid betaclearing/depleting cellular components, an inhibitor of N-terminaltruncated amyloid beta including pyroglutamated amyloid beta 3-42, ananti-inflammatory molecule, a cholinesterase inhibitor, a MI agonist, aamyloid or tau modifying drug, a nutritive supplement, a memantineinhibitor, and a glutaminyl cyclase inhibitor.
 48. A method of treatmentof amyloidosis which comprises administering, to a subject in need ofsuch treatment a therapeutically effective amount of a humanizedantibody of claim
 1. 49. The method according to claim 48 wherein theadministered amount is sufficient to reverse cognitive decline, improvecognition, or prevent cognitive decline in a subject diagnosed with mildcognitive impairment (MCI), Alzheimer's disease (AD), sporadicAlzheimer's disease (SAD), Familial Alzheimer's dementia (FAD),neurodegeneration in Down Syndrome, or cerebral amyloid angiopathy. 50.The method according to claim 48 wherein the administered amount issufficient to remove Aβ N3pE from plaques or other biological complexes.51. The method according to claim 48 wherein the administered amount issufficient to reduce plaque load of affected tissue.
 52. A method ofdetecting an amyloid-associated condition which comprises the steps ofobtaining a biological sample from a patient; contacting the sample witha humanized antibody of claim 1; and detecting an amount of saidhumanized antibody bound to an amyloid protein.
 53. A diagnostic kitwhich comprises a humanized antibody in accordance with claim 1 togetherwith instructions for performing a diagnostic assay of a patient'sbiological sample.
 54. The diagnostic kit in accordance with claim 53wherein the humanized antibody is labeled.